AU2008200027A1 - Evolving new molecular function - Google Patents
Evolving new molecular function Download PDFInfo
- Publication number
- AU2008200027A1 AU2008200027A1 AU2008200027A AU2008200027A AU2008200027A1 AU 2008200027 A1 AU2008200027 A1 AU 2008200027A1 AU 2008200027 A AU2008200027 A AU 2008200027A AU 2008200027 A AU2008200027 A AU 2008200027A AU 2008200027 A1 AU2008200027 A1 AU 2008200027A1
- Authority
- AU
- Australia
- Prior art keywords
- dna
- library
- template
- nucleic acid
- synthesis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000004879 molecular function Effects 0.000 title description 8
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 164
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 155
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 154
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 151
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 151
- 238000000034 method Methods 0.000 claims abstract description 142
- 150000003384 small molecules Chemical class 0.000 claims abstract description 113
- 229920000642 polymer Polymers 0.000 claims abstract description 110
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 62
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 61
- 238000006243 chemical reaction Methods 0.000 claims description 197
- 150000001875 compounds Chemical class 0.000 claims description 93
- 239000000178 monomer Substances 0.000 claims description 84
- 230000027455 binding Effects 0.000 claims description 66
- 108020004566 Transfer RNA Proteins 0.000 claims description 55
- 125000003729 nucleotide group Chemical group 0.000 claims description 54
- 238000003752 polymerase chain reaction Methods 0.000 claims description 53
- 239000002773 nucleotide Substances 0.000 claims description 49
- 239000000376 reactant Substances 0.000 claims description 46
- 238000012546 transfer Methods 0.000 claims description 46
- 230000000694 effects Effects 0.000 claims description 37
- 108020005098 Anticodon Proteins 0.000 claims description 36
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 26
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 26
- 239000000126 substance Substances 0.000 claims description 26
- 238000000338 in vitro Methods 0.000 claims description 24
- 239000000872 buffer Substances 0.000 claims description 21
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 20
- 230000003197 catalytic effect Effects 0.000 claims description 17
- 239000007795 chemical reaction product Substances 0.000 claims description 15
- 238000009396 hybridization Methods 0.000 claims description 15
- 108091008146 restriction endonucleases Proteins 0.000 claims description 7
- 238000002744 homologous recombination Methods 0.000 claims description 3
- 230000006801 homologous recombination Effects 0.000 claims description 3
- GJVCJFPKACWMSC-UHFFFAOYSA-N anticodon Chemical compound O=C1NC(=O)C(C)=CN1C1OC(COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)CO)C(OP(O)(=S)OCC2C(CC(O2)N2C3=C(C(NC(N)=N3)=O)N=C2)O)C1 GJVCJFPKACWMSC-UHFFFAOYSA-N 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 25
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 25
- 229920001222 biopolymer Polymers 0.000 abstract description 22
- 239000000203 mixture Substances 0.000 abstract description 16
- 230000002194 synthesizing effect Effects 0.000 abstract description 13
- 102000040430 polynucleotide Human genes 0.000 abstract description 8
- 108091033319 polynucleotide Proteins 0.000 abstract description 8
- 239000002157 polynucleotide Substances 0.000 abstract description 8
- 108020004414 DNA Proteins 0.000 description 172
- 239000003153 chemical reaction reagent Substances 0.000 description 129
- 239000000047 product Substances 0.000 description 106
- 239000003054 catalyst Substances 0.000 description 77
- 239000002585 base Substances 0.000 description 74
- 125000005647 linker group Chemical group 0.000 description 73
- 108020004705 Codon Proteins 0.000 description 71
- 108091034117 Oligonucleotide Proteins 0.000 description 52
- 238000006116 polymerization reaction Methods 0.000 description 50
- 238000013459 approach Methods 0.000 description 49
- 229910052751 metal Inorganic materials 0.000 description 43
- 239000002184 metal Substances 0.000 description 43
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 42
- -1 3-methyl Sadenosine Chemical compound 0.000 description 42
- 239000003446 ligand Substances 0.000 description 42
- 238000005859 coupling reaction Methods 0.000 description 39
- 238000010168 coupling process Methods 0.000 description 37
- 230000008878 coupling Effects 0.000 description 36
- 239000000758 substrate Substances 0.000 description 36
- 239000011324 bead Substances 0.000 description 32
- 102000006495 integrins Human genes 0.000 description 32
- 108010044426 integrins Proteins 0.000 description 32
- 238000007792 addition Methods 0.000 description 31
- 108090000765 processed proteins & peptides Proteins 0.000 description 29
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 28
- 238000003776 cleavage reaction Methods 0.000 description 27
- 230000007017 scission Effects 0.000 description 27
- 230000014616 translation Effects 0.000 description 27
- 150000001412 amines Chemical class 0.000 description 26
- 150000001413 amino acids Chemical class 0.000 description 26
- 239000001226 triphosphate Substances 0.000 description 26
- 235000011178 triphosphate Nutrition 0.000 description 26
- 238000013519 translation Methods 0.000 description 24
- 238000006664 bond formation reaction Methods 0.000 description 23
- 229960002685 biotin Drugs 0.000 description 22
- 239000011616 biotin Substances 0.000 description 22
- 235000020958 biotin Nutrition 0.000 description 21
- 230000000295 complement effect Effects 0.000 description 21
- 108010090804 Streptavidin Proteins 0.000 description 20
- 239000013615 primer Substances 0.000 description 19
- 238000005755 formation reaction Methods 0.000 description 18
- 108010074860 Factor Xa Proteins 0.000 description 16
- 150000001336 alkenes Chemical class 0.000 description 16
- 238000010976 amide bond formation reaction Methods 0.000 description 16
- 230000000875 corresponding effect Effects 0.000 description 16
- 125000000524 functional group Chemical group 0.000 description 16
- 239000011347 resin Substances 0.000 description 16
- 229920005989 resin Polymers 0.000 description 16
- 150000002148 esters Chemical class 0.000 description 15
- 239000012038 nucleophile Substances 0.000 description 15
- 239000003960 organic solvent Substances 0.000 description 15
- 125000002827 triflate group Chemical group FC(S(=O)(=O)O*)(F)F 0.000 description 15
- 108091028043 Nucleic acid sequence Proteins 0.000 description 14
- 108091093037 Peptide nucleic acid Proteins 0.000 description 14
- 239000002253 acid Substances 0.000 description 14
- 230000002068 genetic effect Effects 0.000 description 14
- 238000000746 purification Methods 0.000 description 14
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 13
- 238000003556 assay Methods 0.000 description 13
- 238000006555 catalytic reaction Methods 0.000 description 13
- 230000003993 interaction Effects 0.000 description 13
- 238000005580 one pot reaction Methods 0.000 description 13
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 13
- 229920002635 polyurethane Polymers 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 12
- 150000003141 primary amines Chemical class 0.000 description 12
- 125000006239 protecting group Chemical group 0.000 description 12
- 108091006629 SLC13A2 Proteins 0.000 description 11
- 230000001965 increasing effect Effects 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 11
- 229920005615 natural polymer Polymers 0.000 description 11
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- 102000053602 DNA Human genes 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 10
- 108010067902 Peptide Library Proteins 0.000 description 10
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 238000007306 functionalization reaction Methods 0.000 description 10
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 10
- 238000004949 mass spectrometry Methods 0.000 description 10
- 150000002739 metals Chemical class 0.000 description 10
- 230000035772 mutation Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 9
- 238000001712 DNA sequencing Methods 0.000 description 9
- 238000006668 aldol addition reaction Methods 0.000 description 9
- 238000000137 annealing Methods 0.000 description 9
- 238000010348 incorporation Methods 0.000 description 9
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 9
- 102000005962 receptors Human genes 0.000 description 9
- 108020003175 receptors Proteins 0.000 description 9
- 231100000241 scar Toxicity 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 150000003573 thiols Chemical class 0.000 description 9
- HDZZVAMISRMYHH-KCGFPETGSA-N tubercidin Chemical compound C1=CC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HDZZVAMISRMYHH-KCGFPETGSA-N 0.000 description 9
- 108090001008 Avidin Proteins 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 238000007295 Wittig olefination reaction Methods 0.000 description 8
- 150000001299 aldehydes Chemical class 0.000 description 8
- 150000001408 amides Chemical class 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 239000005547 deoxyribonucleotide Substances 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 239000007858 starting material Substances 0.000 description 8
- 230000007306 turnover Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- BMTZEAOGFDXDAD-UHFFFAOYSA-M 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium;chloride Chemical compound [Cl-].COC1=NC(OC)=NC([N+]2(C)CCOCC2)=N1 BMTZEAOGFDXDAD-UHFFFAOYSA-M 0.000 description 7
- 239000012736 aqueous medium Substances 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 150000007942 carboxylates Chemical class 0.000 description 7
- 238000006352 cycloaddition reaction Methods 0.000 description 7
- 239000012039 electrophile Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000007040 multi-step synthesis reaction Methods 0.000 description 7
- 230000006798 recombination Effects 0.000 description 7
- 238000005215 recombination Methods 0.000 description 7
- 238000006268 reductive amination reaction Methods 0.000 description 7
- 239000003774 sulfhydryl reagent Substances 0.000 description 7
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 7
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000007993 MOPS buffer Substances 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- 238000010640 amide synthesis reaction Methods 0.000 description 6
- 125000002619 bicyclic group Chemical group 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 125000003636 chemical group Chemical group 0.000 description 6
- 238000010511 deprotection reaction Methods 0.000 description 6
- 238000006077 hetero Diels-Alder cycloaddition reaction Methods 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 231100000350 mutagenesis Toxicity 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 235000021317 phosphate Nutrition 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 6
- 125000006850 spacer group Chemical group 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000007704 transition Effects 0.000 description 6
- 238000006736 Huisgen cycloaddition reaction Methods 0.000 description 5
- 238000006445 Nitro-Michael reaction Methods 0.000 description 5
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 5
- 108020004682 Single-Stranded DNA Proteins 0.000 description 5
- 108010006785 Taq Polymerase Proteins 0.000 description 5
- 108090000190 Thrombin Proteins 0.000 description 5
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- 239000012190 activator Substances 0.000 description 5
- 150000003838 adenosines Chemical class 0.000 description 5
- 125000003277 amino group Chemical group 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 5
- 239000012148 binding buffer Substances 0.000 description 5
- 150000004657 carbamic acid derivatives Chemical class 0.000 description 5
- 150000001768 cations Chemical class 0.000 description 5
- 125000004122 cyclic group Chemical group 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- AFOSIXZFDONLBT-UHFFFAOYSA-N divinyl sulfone Chemical class C=CS(=O)(=O)C=C AFOSIXZFDONLBT-UHFFFAOYSA-N 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000003379 elimination reaction Methods 0.000 description 5
- 150000002576 ketones Chemical class 0.000 description 5
- 239000002777 nucleoside Substances 0.000 description 5
- 125000003835 nucleoside group Chemical group 0.000 description 5
- 125000002524 organometallic group Chemical group 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 229920003023 plastic Polymers 0.000 description 5
- 239000004033 plastic Substances 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 238000006722 reduction reaction Methods 0.000 description 5
- 238000007152 ring opening metathesis polymerisation reaction Methods 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 229960004072 thrombin Drugs 0.000 description 5
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 5
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 5
- 229940045145 uridine Drugs 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- BQWBEDSJTMWJAE-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[(2-iodoacetyl)amino]benzoate Chemical compound C1=CC(NC(=O)CI)=CC=C1C(=O)ON1C(=O)CCC1=O BQWBEDSJTMWJAE-UHFFFAOYSA-N 0.000 description 4
- 108020004635 Complementary DNA Proteins 0.000 description 4
- 238000010485 C−C bond formation reaction Methods 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000007341 Heck reaction Methods 0.000 description 4
- 239000007987 MES buffer Substances 0.000 description 4
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 238000005865 alkene metathesis reaction Methods 0.000 description 4
- 150000001500 aryl chlorides Chemical class 0.000 description 4
- 238000010504 bond cleavage reaction Methods 0.000 description 4
- 238000010804 cDNA synthesis Methods 0.000 description 4
- 150000001735 carboxylic acids Chemical class 0.000 description 4
- 150000001780 cephalosporins Chemical class 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000008030 elimination Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 125000000623 heterocyclic group Chemical group 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000000269 nucleophilic effect Effects 0.000 description 4
- 238000006053 organic reaction Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 4
- LILXDMFJXYAKMK-UHFFFAOYSA-N 2-bromo-1,1-diethoxyethane Chemical compound CCOC(CBr)OCC LILXDMFJXYAKMK-UHFFFAOYSA-N 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 3
- KXDHJXZQYSOELW-UHFFFAOYSA-N Carbamic acid Chemical group NC(O)=O KXDHJXZQYSOELW-UHFFFAOYSA-N 0.000 description 3
- 108010017826 DNA Polymerase I Proteins 0.000 description 3
- 102000004594 DNA Polymerase I Human genes 0.000 description 3
- 230000006820 DNA synthesis Effects 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010049003 Fibrinogen Proteins 0.000 description 3
- 102000008946 Fibrinogen Human genes 0.000 description 3
- 238000006842 Henry reaction Methods 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 108091028664 Ribonucleotide Proteins 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005575 aldol reaction Methods 0.000 description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 150000001414 amino alcohols Chemical class 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 239000011203 carbon fibre reinforced carbon Substances 0.000 description 3
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 3
- 239000002738 chelating agent Substances 0.000 description 3
- 238000006757 chemical reactions by type Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000006114 decarboxylation reaction Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000000326 densiometry Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000007876 drug discovery Methods 0.000 description 3
- 229940012952 fibrinogen Drugs 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 238000007429 general method Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 150000003833 nucleoside derivatives Chemical class 0.000 description 3
- 238000006384 oligomerization reaction Methods 0.000 description 3
- 235000013406 prebiotics Nutrition 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 239000002336 ribonucleotide Substances 0.000 description 3
- 125000002652 ribonucleotide group Chemical group 0.000 description 3
- JXOHGGNKMLTUBP-HSUXUTPPSA-N shikimic acid Chemical compound O[C@@H]1CC(C(O)=O)=C[C@@H](O)[C@H]1O JXOHGGNKMLTUBP-HSUXUTPPSA-N 0.000 description 3
- JXOHGGNKMLTUBP-JKUQZMGJSA-N shikimic acid Natural products O[C@@H]1CC(C(O)=O)=C[C@H](O)[C@@H]1O JXOHGGNKMLTUBP-JKUQZMGJSA-N 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 230000002269 spontaneous effect Effects 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 150000003568 thioethers Chemical class 0.000 description 3
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 238000000844 transformation Methods 0.000 description 3
- XUDGDVPXDYGCTG-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-[2-(2,5-dioxopyrrolidin-1-yl)oxycarbonyloxyethylsulfonyl]ethyl carbonate Chemical compound O=C1CCC(=O)N1OC(=O)OCCS(=O)(=O)CCOC(=O)ON1C(=O)CCC1=O XUDGDVPXDYGCTG-UHFFFAOYSA-N 0.000 description 2
- JKHVDAUOODACDU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCN1C(=O)C=CC1=O JKHVDAUOODACDU-UHFFFAOYSA-N 0.000 description 2
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 2
- XXNHOXKGISCJIY-WOUKDFQISA-N (2r,3r,4s,5r)-2-(6-aminopurin-9-yl)-2-ethenyl-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@]1(C=C)O[C@H](CO)[C@@H](O)[C@H]1O XXNHOXKGISCJIY-WOUKDFQISA-N 0.000 description 2
- NJBIVXMQFIQOGE-KVQBGUIXSA-N (2r,3s,5r)-5-(6-amino-8-bromopurin-9-yl)-2-(hydroxymethyl)oxolan-3-ol Chemical compound BrC1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 NJBIVXMQFIQOGE-KVQBGUIXSA-N 0.000 description 2
- BSSNZUFKXJJCBG-OWOJBTEDSA-N (e)-but-2-enediamide Chemical compound NC(=O)\C=C\C(N)=O BSSNZUFKXJJCBG-OWOJBTEDSA-N 0.000 description 2
- 150000000180 1,2-diols Chemical class 0.000 description 2
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical class C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 2
- NRKYWOKHZRQRJR-UHFFFAOYSA-N 2,2,2-trifluoroacetamide Chemical group NC(=O)C(F)(F)F NRKYWOKHZRQRJR-UHFFFAOYSA-N 0.000 description 2
- LWTIGYSPAXKMDG-UHFFFAOYSA-N 2,3-dihydro-1h-imidazole Chemical compound C1NC=CN1 LWTIGYSPAXKMDG-UHFFFAOYSA-N 0.000 description 2
- HSJKGGMUJITCBW-UHFFFAOYSA-N 3-hydroxybutanal Chemical compound CC(O)CC=O HSJKGGMUJITCBW-UHFFFAOYSA-N 0.000 description 2
- QCPFFGGFHNZBEP-UHFFFAOYSA-N 4,5,6,7-tetrachloro-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 QCPFFGGFHNZBEP-UHFFFAOYSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- ZAYHVCMSTBRABG-JXOAFFINSA-N 5-methylcytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZAYHVCMSTBRABG-JXOAFFINSA-N 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- HDZZVAMISRMYHH-UHFFFAOYSA-N 9beta-Ribofuranosyl-7-deazaadenin Natural products C1=CC=2C(N)=NC=NC=2N1C1OC(CO)C(O)C1O HDZZVAMISRMYHH-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- JPRWMFLCIVUTMH-UHFFFAOYSA-N C(C)OC(COC(CC#N)=O)OCC Chemical compound C(C)OC(COC(CC#N)=O)OCC JPRWMFLCIVUTMH-UHFFFAOYSA-N 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 239000008001 CAPS buffer Substances 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 229930186147 Cephalosporin Natural products 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 108020004394 Complementary RNA Proteins 0.000 description 2
- 108010069514 Cyclic Peptides Proteins 0.000 description 2
- 102000001189 Cyclic Peptides Human genes 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- 108020001019 DNA Primers Proteins 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 229940123583 Factor Xa inhibitor Drugs 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 2
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 2
- MBYJMFIRGBGDHP-KQYNXXCUSA-N I[N+]1=CN([C@H]2[C@H](O)[C@H](O)[C@@H](CO)O2)C=2N=CN=C(C1=2)N Chemical compound I[N+]1=CN([C@H]2[C@H](O)[C@H](O)[C@@H](CO)O2)C=2N=CN=C(C1=2)N MBYJMFIRGBGDHP-KQYNXXCUSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241000872931 Myoporum sandwicense Species 0.000 description 2
- LQZMLBORDGWNPD-UHFFFAOYSA-N N-iodosuccinimide Chemical compound IN1C(=O)CCC1=O LQZMLBORDGWNPD-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229910021120 PdC12 Inorganic materials 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 229920002396 Polyurea Polymers 0.000 description 2
- 101000781681 Protobothrops flavoviridis Disintegrin triflavin Proteins 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 108010022999 Serine Proteases Proteins 0.000 description 2
- 102000012479 Serine Proteases Human genes 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 238000003477 Sonogashira cross-coupling reaction Methods 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 102100031013 Transgelin Human genes 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 238000007239 Wittig reaction Methods 0.000 description 2
- FPKBIYKAYFGTKG-UHFFFAOYSA-N [5-(2-amino-6-oxo-1H-purin-9-yl)-2-[[[2-[[[2-[[[2-[[[2-[[[2-[[[2-[[[2-[[[2-[[[2-[[[2-[[[5-(2-amino-6-oxo-1H-purin-9-yl)-2-[[[5-(2-amino-6-oxo-1H-purin-9-yl)-2-[[[2-[[[2-[[[5-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(6-aminopurin-9-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphoryl]oxymethyl]oxolan-3-yl] [3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methyl hydrogen phosphate Chemical group Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c3nc(N)[nH]c4=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O FPKBIYKAYFGTKG-UHFFFAOYSA-N 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 239000003377 acid catalyst Substances 0.000 description 2
- 239000012445 acidic reagent Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 125000000746 allylic group Chemical group 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 150000001503 aryl iodides Chemical class 0.000 description 2
- 230000003190 augmentative effect Effects 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 229940124587 cephalosporin Drugs 0.000 description 2
- 150000001805 chlorine compounds Chemical class 0.000 description 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- APEJMQOBVMLION-UHFFFAOYSA-N cinnamamide Chemical compound NC(=O)C=CC1=CC=CC=C1 APEJMQOBVMLION-UHFFFAOYSA-N 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 229940104302 cytosine Drugs 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000005595 deprotonation Effects 0.000 description 2
- 238000010537 deprotonation reaction Methods 0.000 description 2
- 238000005828 desilylation reaction Methods 0.000 description 2
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 230000003028 elevating effect Effects 0.000 description 2
- 238000006735 epoxidation reaction Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 150000002170 ethers Chemical class 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 238000012248 genetic selection Methods 0.000 description 2
- 108010064365 glycyl- arginyl-glycyl-aspartyl-seryl-prolyl-lysine Proteins 0.000 description 2
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 150000002466 imines Chemical class 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical group NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 238000005710 macrocyclization reaction Methods 0.000 description 2
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 2
- 238000005649 metathesis reaction Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 231100000243 mutagenic effect Toxicity 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 125000004971 nitroalkyl group Chemical group 0.000 description 2
- 238000010653 organometallic reaction Methods 0.000 description 2
- 210000002997 osteoclast Anatomy 0.000 description 2
- 150000008300 phosphoramidites Chemical class 0.000 description 2
- 238000011907 photodimerization Methods 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 229920000728 polyester Polymers 0.000 description 2
- 239000002685 polymerization catalyst Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000012041 precatalyst Substances 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 238000000159 protein binding assay Methods 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 239000002464 receptor antagonist Substances 0.000 description 2
- 229940044551 receptor antagonist Drugs 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 229910052707 ruthenium Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 238000005556 structure-activity relationship Methods 0.000 description 2
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000001447 template-directed synthesis Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 150000007970 thio esters Chemical class 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 229910052718 tin Inorganic materials 0.000 description 2
- WJKHJLXJJJATHN-UHFFFAOYSA-N triflic anhydride Chemical compound FC(F)(F)S(=O)(=O)OS(=O)(=O)C(F)(F)F WJKHJLXJJJATHN-UHFFFAOYSA-N 0.000 description 2
- 125000002348 vinylic group Chemical group 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- SWHAGWLVMRLFKO-UHFFFAOYSA-N (2-nitrophenyl)methyl carbamate Chemical compound NC(=O)OCC1=CC=CC=C1[N+]([O-])=O SWHAGWLVMRLFKO-UHFFFAOYSA-N 0.000 description 1
- BWGMWFHXKDRHEB-UHFFFAOYSA-N (2-nitrophenyl)methylcarbamic acid Chemical class OC(=O)NCC1=CC=CC=C1[N+]([O-])=O BWGMWFHXKDRHEB-UHFFFAOYSA-N 0.000 description 1
- ZRVZOBGMZWVJOS-VMXHOPILSA-N (2s)-6-amino-2-[[(2s)-1-[(2s)-2-[[(2s)-2-[[2-[[(2s)-2-[(2-aminoacetyl)amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carbonyl]amino]hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)CN ZRVZOBGMZWVJOS-VMXHOPILSA-N 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- XMKLTEGSALONPH-UHFFFAOYSA-N 1,2,4,5-tetrazinane-3,6-dione Chemical compound O=C1NNC(=O)NN1 XMKLTEGSALONPH-UHFFFAOYSA-N 0.000 description 1
- GJFNRSDCSTVPCJ-UHFFFAOYSA-N 1,8-bis(dimethylamino)naphthalene Chemical compound C1=CC(N(C)C)=C2C(N(C)C)=CC=CC2=C1 GJFNRSDCSTVPCJ-UHFFFAOYSA-N 0.000 description 1
- PISWNSOQFZRVJK-XLPZGREQSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methyl-2-sulfanylidenepyrimidin-4-one Chemical compound S=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 PISWNSOQFZRVJK-XLPZGREQSA-N 0.000 description 1
- HRSBDXXXHGAUJF-QJPTWQEYSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-pent-1-ynylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C#CCCC)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 HRSBDXXXHGAUJF-QJPTWQEYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- XBNGYFFABRKICK-UHFFFAOYSA-N 2,3,4,5,6-pentafluorophenol Chemical compound OC1=C(F)C(F)=C(F)C(F)=C1F XBNGYFFABRKICK-UHFFFAOYSA-N 0.000 description 1
- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 description 1
- 150000003923 2,5-pyrrolediones Chemical class 0.000 description 1
- ZDTFMPXQUSBYRL-UUOKFMHZSA-N 2-Aminoadenosine Chemical compound C12=NC(N)=NC(N)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O ZDTFMPXQUSBYRL-UUOKFMHZSA-N 0.000 description 1
- SLTGLTLBIVDQKE-UHFFFAOYSA-N 2-amino-3-(2-aminoethoxy)propanoic acid Chemical compound NCCOCC(N)C(O)=O SLTGLTLBIVDQKE-UHFFFAOYSA-N 0.000 description 1
- JRYMOPZHXMVHTA-DAGMQNCNSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=CC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JRYMOPZHXMVHTA-DAGMQNCNSA-N 0.000 description 1
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 description 1
- MYHNUQVKTSTVDI-UHFFFAOYSA-N 2-methyl-2-[(2-methylpropan-2-yl)oxy]propane;potassium Chemical compound [K].CC(C)(C)OC(C)(C)C MYHNUQVKTSTVDI-UHFFFAOYSA-N 0.000 description 1
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- RHFUOMFWUGWKKO-XVFCMESISA-N 2-thiocytidine Chemical compound S=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RHFUOMFWUGWKKO-XVFCMESISA-N 0.000 description 1
- OBJOREYUKFGGKX-UHFFFAOYSA-N 3-[2-[3-(4-methylpiperazin-1-yl)anilino]pyrrolo[2,3-d]pyrimidin-7-yl]benzamide Chemical compound C1CN(C)CCN1C1=CC=CC(NC=2N=C3N(C=4C=C(C=CC=4)C(N)=O)C=CC3=CN=2)=C1 OBJOREYUKFGGKX-UHFFFAOYSA-N 0.000 description 1
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical compound OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 description 1
- XXSIICQLPUAUDF-TURQNECASA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-prop-1-ynylpyrimidin-2-one Chemical compound O=C1N=C(N)C(C#CC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 XXSIICQLPUAUDF-TURQNECASA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- ASYBZHICIMVQII-UHFFFAOYSA-N 4-hydroxylysine Chemical compound NCCC(O)CC(N)C(O)=O ASYBZHICIMVQII-UHFFFAOYSA-N 0.000 description 1
- ZAYHVCMSTBRABG-UHFFFAOYSA-N 5-Methylcytidine Natural products O=C1N=C(N)C(C)=CN1C1C(O)C(O)C(CO)O1 ZAYHVCMSTBRABG-UHFFFAOYSA-N 0.000 description 1
- AGFIRQJZCNVMCW-UAKXSSHOSA-N 5-bromouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 AGFIRQJZCNVMCW-UAKXSSHOSA-N 0.000 description 1
- FHIDNBAQOFJWCA-UAKXSSHOSA-N 5-fluorouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 FHIDNBAQOFJWCA-UAKXSSHOSA-N 0.000 description 1
- KDOPAZIWBAHVJB-UHFFFAOYSA-N 5h-pyrrolo[3,2-d]pyrimidine Chemical compound C1=NC=C2NC=CC2=N1 KDOPAZIWBAHVJB-UHFFFAOYSA-N 0.000 description 1
- UEHOMUNTZPIBIL-UUOKFMHZSA-N 6-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-7h-purin-8-one Chemical compound O=C1NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O UEHOMUNTZPIBIL-UUOKFMHZSA-N 0.000 description 1
- HSHGZXNAXBPPDL-HZGVNTEJSA-N 7beta-aminocephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H]([NH3+])[C@@H]12 HSHGZXNAXBPPDL-HZGVNTEJSA-N 0.000 description 1
- HCAJQHYUCKICQH-VPENINKCSA-N 8-Oxo-7,8-dihydro-2'-deoxyguanosine Chemical compound C1=2NC(N)=NC(=O)C=2NC(=O)N1[C@H]1C[C@H](O)[C@@H](CO)O1 HCAJQHYUCKICQH-VPENINKCSA-N 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- 101710134784 Agnoprotein Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 108010031480 Artificial Receptors Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- RENMDAKOXSCIGH-UHFFFAOYSA-N Chloroacetonitrile Chemical compound ClCC#N RENMDAKOXSCIGH-UHFFFAOYSA-N 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- XPYBSIWDXQFNMH-UHFFFAOYSA-N D-fructose 1,6-bisphosphate Natural products OP(=O)(O)OCC(O)C(O)C(O)C(=O)COP(O)(O)=O XPYBSIWDXQFNMH-UHFFFAOYSA-N 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010069941 DNA receptor Proteins 0.000 description 1
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 description 1
- 108091027757 Deoxyribozyme Proteins 0.000 description 1
- 238000006117 Diels-Alder cycloaddition reaction Methods 0.000 description 1
- 238000005698 Diels-Alder reaction Methods 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 208000005331 Hepatitis D Diseases 0.000 description 1
- 208000037262 Hepatitis delta Diseases 0.000 description 1
- 108010007267 Hirudins Proteins 0.000 description 1
- 102000007625 Hirudins Human genes 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- GHSJKUNUIHUPDF-BYPYZUCNSA-N L-thialysine Chemical compound NCCSC[C@H](N)C(O)=O GHSJKUNUIHUPDF-BYPYZUCNSA-N 0.000 description 1
- 239000002841 Lewis acid Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 238000006683 Mannich reaction Methods 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 238000006845 Michael addition reaction Methods 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- GDIHDSKOVXEJQQ-UHFFFAOYSA-N NC(O)=O.NC(O)=O.NC(O)=O.N Chemical compound NC(O)=O.NC(O)=O.NC(O)=O.N GDIHDSKOVXEJQQ-UHFFFAOYSA-N 0.000 description 1
- 101100028920 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cfp gene Proteins 0.000 description 1
- JQGGAELIYHNDQS-UHFFFAOYSA-N Nic 12 Natural products CC(C=CC(=O)C)c1ccc2C3C4OC4C5(O)CC=CC(=O)C5(C)C3CCc2c1 JQGGAELIYHNDQS-UHFFFAOYSA-N 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108090000279 Peptidyltransferases Proteins 0.000 description 1
- YGYAWVDWMABLBF-UHFFFAOYSA-N Phosgene Chemical class ClC(Cl)=O YGYAWVDWMABLBF-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 239000007868 Raney catalyst Substances 0.000 description 1
- 229910000564 Raney nickel Inorganic materials 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- 238000007011 Robinson annulation reaction Methods 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229910008066 SnC12 Inorganic materials 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 238000006619 Stille reaction Methods 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 238000006859 Swern oxidation reaction Methods 0.000 description 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 1
- 101000865057 Thermococcus litoralis DNA polymerase Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- RHQDFWAXVIIEBN-UHFFFAOYSA-N Trifluoroethanol Chemical compound OCC(F)(F)F RHQDFWAXVIIEBN-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- DILVWDXOASDRPN-MROZADKFSA-N [(2r,3r,4r)-3,4,5-trihydroxy-1-oxopentan-2-yl] dihydrogen phosphate Chemical group OC[C@@H](O)[C@@H](O)[C@H](C=O)OP(O)(O)=O DILVWDXOASDRPN-MROZADKFSA-N 0.000 description 1
- WYBZFSGKJOYRQH-UHFFFAOYSA-N [nitro(phenyl)methyl] carbamate Chemical compound NC(=O)OC([N+]([O-])=O)C1=CC=CC=C1 WYBZFSGKJOYRQH-UHFFFAOYSA-N 0.000 description 1
- POIOOCHMXHKUHV-UHFFFAOYSA-N [nitro-[nitro(phenyl)methoxy]methyl]benzene Chemical compound C=1C=CC=CC=1C([N+](=O)[O-])OC([N+]([O-])=O)C1=CC=CC=C1 POIOOCHMXHKUHV-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 150000001241 acetals Chemical class 0.000 description 1
- CIHUOJZDKZENCP-UHFFFAOYSA-N acetonitrile;triethylazanium;acetate Chemical compound CC#N.CC(O)=O.CCN(CC)CC CIHUOJZDKZENCP-UHFFFAOYSA-N 0.000 description 1
- 238000007171 acid catalysis Methods 0.000 description 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 150000001253 acrylic acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000005882 aldol condensation reaction Methods 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- 125000005262 alkoxyamine group Chemical group 0.000 description 1
- RNBGYGVWRKECFJ-ZXXMMSQZSA-N alpha-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ZXXMMSQZSA-N 0.000 description 1
- 125000000266 alpha-aminoacyl group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000006229 amino acid addition Effects 0.000 description 1
- IVHKZGYFKJRXBD-UHFFFAOYSA-N amino carbamate Chemical compound NOC(N)=O IVHKZGYFKJRXBD-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000005915 ammonolysis reaction Methods 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000005899 aromatization reaction Methods 0.000 description 1
- 150000001502 aryl halides Chemical class 0.000 description 1
- 125000003288 aryl iodide group Chemical group 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000005815 base catalysis Methods 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 description 1
- 108010055460 bivalirudin Proteins 0.000 description 1
- 229960001500 bivalirudin Drugs 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 230000031709 bromination Effects 0.000 description 1
- 238000005893 bromination reaction Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 150000005829 chemical entities Chemical class 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000007809 chemical reaction catalyst Substances 0.000 description 1
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
- 239000005516 coenzyme A Substances 0.000 description 1
- 229940093530 coenzyme a Drugs 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000007819 coupling partner Substances 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 125000001295 dansyl group Chemical group [H]C1=C([H])C(N(C([H])([H])[H])C([H])([H])[H])=C2C([H])=C([H])C([H])=C(C2=C1[H])S(*)(=O)=O 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 230000027832 depurination Effects 0.000 description 1
- QQJDHWMADUVRDL-UHFFFAOYSA-N didodecyl(dimethyl)azanium Chemical compound CCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCC QQJDHWMADUVRDL-UHFFFAOYSA-N 0.000 description 1
- 150000001993 dienes Chemical class 0.000 description 1
- 238000005906 dihydroxylation reaction Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- JGUQDUKBUKFFRO-CIIODKQPSA-N dimethylglyoxime Chemical compound O/N=C(/C)\C(\C)=N\O JGUQDUKBUKFFRO-CIIODKQPSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- OAEGRYMCJYIXQT-UHFFFAOYSA-N dithiooxamide Chemical compound NC(=S)C(N)=S OAEGRYMCJYIXQT-UHFFFAOYSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- FXPHJTKVWZVEGA-UHFFFAOYSA-N ethenyl hydrogen carbonate Chemical group OC(=O)OC=C FXPHJTKVWZVEGA-UHFFFAOYSA-N 0.000 description 1
- ZIUSEGSNTOUIPT-UHFFFAOYSA-N ethyl 2-cyanoacetate Chemical compound CCOC(=O)CC#N ZIUSEGSNTOUIPT-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 125000005313 fatty acid group Chemical group 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- RNBGYGVWRKECFJ-UHFFFAOYSA-N fructose-1,6-phosphate Natural products OC1C(O)C(O)(COP(O)(O)=O)OC1COP(O)(O)=O RNBGYGVWRKECFJ-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 229940015043 glyoxal Drugs 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 230000020169 heat generation Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 208000029570 hepatitis D virus infection Diseases 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 1
- 229940006607 hirudin Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical group [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 150000001261 hydroxy acids Chemical class 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000011967 lanthanide triflate Substances 0.000 description 1
- 150000007517 lewis acids Chemical class 0.000 description 1
- 238000002898 library design Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 150000002668 lysine derivatives Chemical class 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000006263 metalation reaction Methods 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- WAERZCCNJRHZFS-QXUBKYEPSA-N mismatched pna Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1C(=O)CN(C(=O)CN1C2=C(C(NC(N)=N2)=O)N=C1)CCNC(=O)CN(C(=O)CN1C2=C(C(NC(N)=N2)=O)N=C1)CCNC(=O)CN(C(=O)CN1C2=NC=NC(N)=C2N=C1)CCNC(=O)CN(C(=O)CN1C2=NC=NC(N)=C2N=C1)CCNC(=O)CN(C(=O)CN1C(NC(=O)C(C)=C1)=O)CCNC(=O)CN(C(=O)CN1C2=NC=NC(N)=C2N=C1)CCNC(=O)CN(C(=O)CN1C2=C(C(NC(N)=N2)=O)N=C1)CCNC(=O)CN(C(=O)CN1C(N=C(N)C=C1)=O)CCNC(=O)CN(C(=O)CN1C2=NC=NC(N)=C2N=C1)CCNC(=O)CN(C(=O)CN1C2=NC=NC(N)=C2N=C1)CCNC(=O)CN(C(=O)CN1C2=C(C(NC(N)=N2)=O)N=C1)CCNC(=O)CN(C(=O)CN1C(NC(=O)C(C)=C1)=O)CCNC(=O)CN(C(=O)CN1C(NC(=O)C(C)=C1)=O)CCNC(=O)CN(C(=O)CN1C(NC(=O)C(C)=C1)=O)CCNC(=O)CN(CCN)C(=O)CN1C(=O)NC(=O)C(C)=C1 WAERZCCNJRHZFS-QXUBKYEPSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- SQDFHQJTAWCFIB-UHFFFAOYSA-N n-methylidenehydroxylamine Chemical compound ON=C SQDFHQJTAWCFIB-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 150000002902 organometallic compounds Chemical class 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- IZUPBVBPLAPZRR-UHFFFAOYSA-N pentachloro-phenol Natural products OC1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl IZUPBVBPLAPZRR-UHFFFAOYSA-N 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 150000003003 phosphines Chemical class 0.000 description 1
- 125000005541 phosphonamide group Chemical group 0.000 description 1
- SWFDTFBWXCNRGN-UHFFFAOYSA-N phosphonato phosphate;tributylazanium Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O.CCCC[NH+](CCCC)CCCC.CCCC[NH+](CCCC)CCCC.CCCC[NH+](CCCC)CCCC.CCCC[NH+](CCCC)CCCC SWFDTFBWXCNRGN-UHFFFAOYSA-N 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical group 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920001281 polyalkylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229930001119 polyketide Natural products 0.000 description 1
- 125000000830 polyketide group Chemical group 0.000 description 1
- 229920000307 polymer substrate Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- RHFUOMFWUGWKKO-UHFFFAOYSA-N s2C Natural products S=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 RHFUOMFWUGWKKO-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000003333 secondary alcohols Chemical class 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- KZJPVUDYAMEDRM-UHFFFAOYSA-M silver;2,2,2-trifluoroacetate Chemical compound [Ag+].[O-]C(=O)C(F)(F)F KZJPVUDYAMEDRM-UHFFFAOYSA-M 0.000 description 1
- 238000006884 silylation reaction Methods 0.000 description 1
- 230000015590 smooth muscle cell migration Effects 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000006104 solid solution Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- FGTJJHCZWOVVNH-UHFFFAOYSA-N tert-butyl-[tert-butyl(dimethyl)silyl]oxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)O[Si](C)(C)C(C)(C)C FGTJJHCZWOVVNH-UHFFFAOYSA-N 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 125000005413 thiopyridyl group Chemical group 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 238000007070 tosylation reaction Methods 0.000 description 1
- 229940043263 traditional drug Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- XQKBFQXWZCFNFF-UHFFFAOYSA-K triiodosamarium Chemical compound I[Sm](I)I XQKBFQXWZCFNFF-UHFFFAOYSA-K 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- WVLBCYQITXONBZ-UHFFFAOYSA-N trimethyl phosphate Chemical compound COP(=O)(OC)OC WVLBCYQITXONBZ-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 150000003672 ureas Chemical class 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polyesters Or Polycarbonates (AREA)
- Steroid Compounds (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
- Peptides Or Proteins (AREA)
Abstract
Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The present invention provides methods, compositions, and systems for synthesizing, selecting, amplifying, and evolving non-natural molecules based on nucleic acid templates. The sequence of a nucleic acid template is used to direct the synthesis of non-natural molecules such as unnatural polymers and small molecules. Using this method combinatorial libraries of these molecules can be prepared and screened. Upon selection of a molecule, its encoding nucleic acid template may be amplified and/or evolved to yield the same molecule of the present invention allow for the amplification and evolution of non-natural molecules in a manner analogues to the amplification of natural biopolymer such as polynucleotides and protein.
Description
Regulation 3.2
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT
APPLICANT:
Invention Title: PRESIDENT AND FELLOWS OF HARVARD COLLEGE EVOLVING NEW MOLECULAR FUNCTION The following statement is a full description of this invention, including the best method of performing it known to me:
I
WO 02/074929 PCT/UIS02/08546 00 O EVOLVING NEW MOLECULAR FUNCTION SPRIORITY INFORMATION (0001] This application claims priority under 35 U.S.C. 119(e) to U.S. Provisional patent applications 60/277,081, filed March 19, 2001, entitled "Nucleic Acid Directed Synthesis of Chemical Compounds" 60/277,094, filed March 19, 2001, entitled "Approaches to Generating 0New Molecular Function"; and 60/306,691, filed July 20, 2001, entitled "Approaches to Generating New Molecular Function", and the entire contents of each of these applications are 00 hereby incorporated by reference.
C
BACKGROUND OF THE INVENTION [00021 The classic "chemical approach" to generating molecules with new functions has been used extensively over the last century in applications ranging from drug discovery to synthetic methodology to materials science. In this approach (Fig. 1, black), researchers synthesize or isolate candidate molecules, assay these candidates for desired properties, determine the structures of active compounds if unknown, formulate structure-activity relationships based on the assay and structural data, and then synthesize a new generation of molecules designed to possess improved properties. While combinatorial chemistry methods (see, for example, A. V.
Eliseev and J. M. Lehn. Combinatorial Chemistry In Biology 1999, 243, 159-172; K. W. Kuntz, M. L. Snapper and A. H. Hoveyda. Current Opinion in Chemical Biology 1999, 3, 313-319; D.
R. Liu and P. G. Schultz. Angew. Chem. Intl. Ed. Eng. 1999, 38, 36) have increased the throughput of this approach, its fundamental limitations remain unchanged. Several factors limit the effectiveness of the chemical approach to generating molecular function. First, our ability to accurately predict the structural changes that will lead to new function is often inadequate due to subtle conformational rearrangements of molecules, unforeseen solvent interactions, or unknown stereochemical requirements of binding or reaction events. The resulting complexity of structure-activity relationships frequently limits the success of rational ligand or catalyst design, including those efforts conducted in a high-throughput manner. Second, the need to assay or screen, rather than select, each member of a collection of candidates limits the number of molecules that can be searched in each experiment. Finally, the lack of a way to amplify synthetic molecules places requirements on the minimum amount of material that must be 1 of 88 WO 02/074929 PCT/US02/08546 00 O produced for characterization, screening, and structure elucidation. As a result, it can be difficult to generate libraries of more than roughly 106 different synthetic compounds.
[0003] In contrast, Nature generates proteins with new functions using a fundamentally C different method that overcomes many of these limitations. In this approach (Fig. 1, gray), a protein with desired properties induces the survival and amplification of the information encoding that protein. This information is diversified through spontaneous mutation and DNA C1 recombination, and then translated into a new generation of candidate proteins using the Sribosome. The power of this process is well appreciated (see, F. Arnold Acc. Chem. Res. 1998, NC 31, 125; F.H. Arnold et al. Curr. Opin. Chem. Biol. 1999, 3, 54-59; J. Minshull et al. Curr. Opin.
00 SChem. Biol. 1999, 3, 284-90) and is evidenced by the fact that proteins and nucleic acids Sdominate the solutions to many complex chemical problems despite their limited chemical functionality. Clearly, unlike the linear chemical approach described above, the steps used by Nature form a cycle of molecular evolution. Proteins emerging from this process have been directly selected, rather than simply screened, for desired activities. Because the information encoding evolving proteins (DNA) can be amplified, a single protein molecule with desired activity can in theory lead to the survival and propagation of the DNA encoding its structure.
The vanishingly small amounts of material needed to participate in a cycle of molecular evolution allow libraries much larger in diversity than those synthesized by chemical approaches to be generated and selected for desired function in small volumes.
[0004] Acknowledging the power and efficiency of Nature's approach, researchers have used molecular evolution to generate many proteins and nucleic acids with novel binding or catalytic properties (see, for example, J. Minshull et al. Curr. Opin. Chem. Biol. 1999, 3, 284-90; C.
Schmidt-Dannert et al. Trends Biotechnol. 1999, 17, 135-6; D. S. Wilson et al. Annu. Rev.
Biochem. 1999, 68, 611-47). Proteins and nucleic acids evolved by researchers have demonstrated value as research tools, diagnostics, industrial reagents, and therapeutics and have greatly expanded our understanding of the molecular interactions that endow proteins and nucleic acids with binding or catalytic properties (see, M. Famulok et al. Curr. Opin. Chem. Biol.
1998, 2, 320-7).
[0005] Despite nature's efficient approach to generating function, nature's molecular evolution is limited to two types of "natural" molecules- proteins and nucleic acids- because thus far the information in DNA can only be translated into proteins or into other nucleic acids.
2 of 88 WO 02/074929 PCT/IlS02/0854(i 00 However, many synthetic molecules of interest do not in general represent nucleic acid backbones, and the use of DNA-templated synthesis to translate DNA sequences into synthetic ct small molecules would be broadly useful only if synthetic molecules other than nucleic acids and Cc nucleic acid analogs could be synthesized in a DNA-templated fashion. An ideal approach to generating functional molecules would merge the most powerful aspects of molecular evolution with the flexibility of synthetic chemistry. Clearly, enabling the evolution of non-natural Csynthetic small molecules and polymers, similarly to the way nature evolves biomolecules, would lead to much more effective methods of discovering new synthetic ligands, receptors, and Scatalysts difficult or impossible to generate using rational design.
00 SUMMARY OF THE INVENTION [0006] The recognition of the need to be able to amplify and evolve classes of molecules besides nucleic acids and proteins led to the present invention providing methods and compositions for the template-directed synthesis, amplification, and evolution of molecules. In general, these methods use an evolvable template to direct the synthesis of a chemical compound or library of chemical compounds the template actually encodes the synthesis of a chemical compound). Based on a library encoded and synthesized using a template such as a nucleic acid, methods are provided for amplifying, evolving, and screening the library. In certain embodiments of special interest, the chemical compounds are compounds that are not, or do not resemble, nucleic acids or analogs thereof. In certain embodiments, the chemical compounds of these template-encoded combinatorial libraries are polymers and more preferably are unnatural polymers excluding natural peptides, proteins, and polynucleotides). In other embodiments, the chemical compounds are small molecules.
[00071 In certain embodiments, the method of synthesizing a compound or library of compounds comprises first providing one or more nucleic acid templates, which one or more nucleic acid templates optionally have a reactive unit associated therewith. The nucleic acid template is then contacted with one or more transfer units designed to have a first moiety, an anti-codon, which hybridizes to a sequence of the nucleic acid, and is associated with a second moiety, a reactive unit, which includes a building block of the compound to be synthesized.
Once these transfer units have hybridized to the nucleic acid template in a sequence-specific manner, the synthesis of the chemical compound can take place due to the interaction of reactive 3 of 88 Tn n~lfi4939 PCT/S02/08546 WO 02/074929Q PCTIUJS02/08546 00 moieties present on the transfer units and/or the nucleic acid template. Signficantly, the Ssequence of the nucleic acid can later be determined to decode the synthetic history of the attached compound and thereby its structure. It will be appreciated that the method described MC) herein may be used to synthesize one molecule at a time or may be used to synthesize thousands to millions of compounds using combinatorial methods.
[0008] It will be appreciated that libraries synthesized in this manner having been CK encoded by a nucleic acid) have the advantage of being amplifiable and evolvable. Once a molecule is identified, its nucleic acid template besides acting as a tag used to identify the C attached compound can also be amplified using standard DNA techniques such as the 00 polymerase chain reaction (PCR). The amplified nucleic acid can then be used to synthesize more of the desired compound. In certain embodiments, during the amplification step mutations are introduced into the nucleic acid in order to generate a population of chemical compounds that are related to the parent compound but are modified at one or more sites. The mutated nucleic acids can then be used to synthesize a new library of related compounds. In this way, the library being screened can be evolved to contain more compounds with the desired activity or to contain compounds with a higher degree of activity.
[0009] The methods of the present invention may be used to synthesize a wide variety of chemical compounds. In certain embodiments, the methods are used to synthesize and evolve unnatural polymers excluding polynucleotides and peptides), which cannot be amplified and evolved using standard techniques currently available. In certain other embodiments, the inventive methods and compositions are utilized for the synthesis of small molecules that are not typically polymeric. In still other embodiments, the method is utilized for the generateion of non-natural nucleic acid polymers.
[0010] The present invention also provides the transfer molecules nucleic acid templates and/or transfer units) useful in the practice of the inventive methods. These transfer molecules typically include a portion capable of hybridizing to a sequence of nucleic acid and a second portion with monomers, other building blocks, or reactants to be incorporated into the final compound being synthesized. It will be appreciated that the two portions of the transfer molecule are preferably associated with each other either directly or through a linker moiety. It will also be appreciated that the reactive unit and the anti-codon may be present in the same molecule a non-natural nucleotide having functionality incorporated therein).
4 of 88 WO 02/074929 PCT/US02/08546 00 [0011] The present invention also provides kits and compositions useful in the practice of the Sinventive methods. These kits may include nucleic acid templates, transfer molecules, C. monomers, solvents, buffers, enzymes, reagents for PCR, nucleotides, small molecule scaffolds, n etc. The kit may be used in the synthesis of a particular type of unnatural polymer or small molecule.
C
DEFINITIONS
S[0012] The term antibody refers to an immunoglobulin, whether natural or wholly or c partially synthetically produced. All derivatives thereof which maintain specific binding ability 00 Sare also included in the term. The term also covers any protein having a binding domain which Cl is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.
[0013] The term, associated with, is used to describe the interaction between or among two or more groups, moieties, compounds, monomers, etc. When two or more entities are "associated with" one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Preferably, the association is covalent. The covalent association may be through an amide, ester, carbon-carbon, disulfide, carbamate, ether, or carbonate linkage.
The covalent association may also include a linker moiety such as a photocleavable linker.
Desirable non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. Also, two or more entities or agents may be "associated" with one another by being present together in the same composition.
[0014] A biological macromolecule is a polynucleotide RNA, DNA, RNA/DNA hybrid), protein, peptide, lipid, natural product, or polysaccharide. The biological macromolecule may be naturally occurring or non-naturally occurring. In a preferred embodiment, a biological macromolecule has a molecular weight greater than 500 g/mol.
[0015] Polynucleotide, nucleic acid, or oligonucleotide refers to a polymer of nucleotides.
The polymer may include natural nucleosides adenosine, thymidine, guanosine, cytidine, of 88 00 WO 02/074929 PCT/US02/08546
O
uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside Sanalogs 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl Sadenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases methylated bases), intercalated bases, modified sugars 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or modified phosphate groups phosphorothioates and 5' -N-phosphoramidite linkages).
.1 [0016] A protein comprises a polymer of amino acid residues linked together by peptide 00 Sbonds. The term, as used herein, refers to proteins, polypeptides, and peptide of any size, C1 structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. A protein may refer to a full-length protein or a fragment of a protein. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, http://www.cco.caltech.edu/~dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be just a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these.
[0017] The term small molecule, as used herein, refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are "natural product-like", however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, a small molecule is typically characterized in that it possesses one or more of the following characteristics including having several carbon-carbon bonds, having multiple stereocenters, having multiple functional groups, having at least two different types of functional groups, and having a molecular weight 6 of 88 00
O
of less than 1500, although this characterization is not intended to be limiting for the purposes of the present invention.
S [0018] The term small molecule scaffold, as used herein, refers to a chemical compound having at least one site for functionalization. In a preferred embodiment, the small molecule S scaffold may have a multitude of sites for functionalization. These functionalization sites may be protected or masked as would be appreciated by one of skill in this art. The sites may also be found on an underlying ring structure or backbone.
00 [0019] The term transfer unit, as used herein, refers to a molecule comprising an anti-codon moiety associated with a reactive unit, including, but not limited to a building block, monomer, monomer unit, or reactant used in synthesizing the nucleic acid-encoded molecules.
DESCRIPTION OF THE FIGURES [0020] Figure 1 depicts nature's approach (gray) and the classical chemical approach (black) to generating molecular function.
[0021] Figures 2A and 2B depict certain DNA-templated reactions for nucleic acids and analogs thereof.
[0022] Figure 3 depicts the general method for synthesizing a polymer using nucleic acidtemplated synthesis.
[0023] Figure 4 shows a quadruplet and triplet non-frameshifting codon set. Each set provides nine possible codons.
[0024] Figure 5 shows methods of screening a library for bond-cleavage and bond-formation catalysts. These methods take advantage of streptavidin's natural affinity for biotin.
[0025] Figure 6A depicts the synthesis directed by hairpin and end-of-helix DNA templates. Reactions were analyzed by denaturing PAGE after the indicated reaction times.
Lanes 3 and 4 contained templates quenched with excess P-mercaptoethanol prior to reaction.
[0026] Figure 6B depicts matched or mismatched reagents linked to thiols or primary amines were mixed with I equiv of template functionalized with the variety of electrophiles shown. Reactions with thiol reagents were conducted at pH 7.5 under the following conditions: SIAB and SBAP: 37°C, 16 h; SIA: 25'C, 16 h, SMCC, GMBS, BMPS, SVSB: 0 C, 10 min. Reactions with amine reagents were conducted at 25 0 C, pH 8.5 for 75 minutes.
7 of 88 00
O
[0027] Figure 7 depicts H templates linked to a-iodoacetamide group which were reacted ct with thiol reagents containing 0, 1, or 3 mismatches at 25°C. Reactions in were repeated C at the indicated temperature for 16 h. Calculated reagent Tm: 38°C (matched), 28 0 C (single mismatch).
[0028] Figure 8 depicts a reaction performed using a 41-base E template and a r reagent designed to anneal 1-30 bases from the 5' end of the template. The kinetic profiles in the graph show the average of two trials (deviations The "n 1 mis" reagent contains three O mismatches.
00 [0029] Figure 9 depicts the repeated n 10 reaction in Figure 8 in which the nine bases following the 5'-NH2-dT were replaced with the backbone analogues shown. Five equivalents of a DNA oligonucleotide complementary to the intervening bases were added to the "DNA clamp" reaction. Reagents were matched or contained three mismatches The gel shows reactions at 25 0 C after 25 min.
[0030] Figure 10 depicts the n 1, n 10, and n 1 mismatched (mis) reactions described in Figure 8 which were repeated with template and reagent concentrations of 12.5, 25, 62.5 or 125 nM.
[0031] Figures 11A and 11B depict a model translation, selection and amplification of synthetic molecules that bind streptavidin from a DNA-encoded library.
[0032] Figure 12A depicts Lanes 1 and 5: PCT: amplified library before streptavidin binding selection. Lanes 2 and 6: PCR amplified library after selection. Lanes 3 and 7: PCR amplified authentic biotin-encoding template. Lane 4: 20 bp ladder. Lanes 5-7 were digested with Tsp45I. DNA sequencing traces of the amplified templates before and after selection are also shown, together with the sequences of the non-biotin encoding and biotin-encoding templates. Figure 12B depicts a general scheme for the creation and evolution of libraries of non-natural molecules using DNA-templated synthesis, where -R 1 represents the library of product functionality transferred from reagent library 1 and -RIB represents a selected product.
[0033] Figures 13A-13D depict exemplary DNA-templated reactions. For all reactions under the specified conditions, product yields of reactions with matched template and reagent sequences were greater than 20-fold higher than that of control reactions with scrambled reagent sequences. Reactions were conducted at 25 0 C with one equivalent each of template and reagent at 60 nM final concentration unless otherwise specified. Conditions: a) 3 mM NaBH 3 CN, 0.1 M 8 of 88 00 MVES buffer pH 6.0, 0.5 M NaCI, 1.5 h; b) 0.1 M TAPS buffer pH 8.5, 300 mM NaC1, 12 h; c) t 0.1 M pH 8.0 TAPS buffer, 1 M NaCI, 5'C, 1.5 h; d) 50 mM MOPS buffer pH 7.5, 2.8 M NaC1, S 22 h; e) 120 nM 19, 1.4 mM Na 2 PdC14, 0.5 M NaOAc buffer pH 5.0, 18 h; f) Premix Na 2 PdC1 4 with two equivalents of P(p-SO 3
C
6
H
4 3 in water 15 min., then add to reactants in 0.5 M NaOAc buffer pH 5.0, 75 mM NaCI, 2 h (final [Pd] 0.3 mM, [19] 120 nM). The olefin geometry of S products from 13 and the regiochemistries of cycloaddition products from 14 and 16 are presumed but not verified.
[0034] Figure 14 depicts analysis by denaturing polyacrylamide gel electrophoresis of representative DNA-templated reactions listed in Figures 13A-13D and 15A and 15B. The structures of reagents and templates correspond to the numbering in Figures 13A-13D, 15A, and Lanes 1, 3, 5, 7, 9, 11: reaction of matched (complementary) reagents and templates under conditions listed in Figures 13A-13D, 15A, and 15B (the reaction of 4 and 6 was mediated by DMT-MM). Lanes 2, 4, 6, 8, 10, 12: reaction of mismatched (non-complementary) reagents and templates under conditions identical to those in lanes 1, 3, 5, 7, 9 and 11, respectively.
[0035] Figures 15A and 15B depict DNA-templated amide bond formation mediated by EDC and sulfo-NHS or by DMT-MM for a variety of substituted carboxylic acids and amines.
In each row, yields of DMT-MM-mediated reactions between reagents and templates complementary in sequence are followed by yields of EDC and sulfo-NHS-mediated reactions.
Conditions: 60 nM template, 120 nM reagent, 50 mM DMT-MM in 0.1 M MOPS buffer pH 1 M NaC1, 16 h, 25 0 C; or 60 nM template, 120 nM reagent, 20 mM EDC, 15 mM sulfo-NHS, 0.1 M MES buffer pH 6.0, 1 M NaC1, 16 h, 25 0 C. In all cases, control reactions with mismatched reagent sequences yielded little or no detectable product.
[0036] Figure 16 depicts Conceptual model for distance-independent DNA-templated synthesis. As the distance between the reactive groups of an annealed reagent and template is increased, the rate of bond formation is presumed to decrease. For those values of n in which the rate of bond formation is significantly higher than the rate of template-reagent annealing, the rate of product formation remains constant. In this regime, the DNA-templated reaction shows distance independence. Denaturing polyacrylamide gel electrophoresis of a DNA-templated Wittig olefination between complementary 11 and 13 with either zero bases (lanes 1-3) or ten bases (lanes 4-6) separating annealed reactants. Although the apparent second order rate constants for the n 0 and n 10 reactions differ by three-fold (kapp (n 0) 9.9 x 103 M-s' l 9 of 88 ir/\ m^ ~~mrrrnr\~ mnrlr 00 wU VVU/U/ IL4Y PCI/USU2/0.34 Owhile kapp (n 10) 3.5 x 103 product yields after 13 h at both distances are nearly Squantitative. Control reactions containing sequence mismatches yielded no detectable product (not shown).
[0037] Figure 17 depicts certain exemplary DNA-templated complexity building reactions.
[0038] Figure 18 depicts certain exemplary linkers for use in the method of the invention.
[0039] Figure 19 depicts certain additional exemplary linkers for use in the method of the invention.
[0040] Figure 20 depicts an exemplary thioester linker for use in the method of the Sinvention.
00 0 [0041] Figure 21 depicts DNA-templated amide bond formation reactions in which reagents C and templates are complexed with dimethyldidodecylammonium cations.
[0042] Figure 22 depicts the assembly of transfer units along the nucleic acid template and polymerization of the nucleotide anti-codon moieties.
[0043] Figure 23 depicts the polymerization of the dicarbamate units along the nucleic acid template to form a polycarbamate. To initiate polymerization the "start" monomer ending in a onitrobenzylcarbamate is photodeprotected to reveal the primary amine that initiates carbamate polymerization. Polymerization then proceeds in the 5' to 3' direction along the DNA backbone, with each nucleophilic attack resulting in the subsequent unmasking of a new amine nucleophile.
Attack of the "stop" monomer liberates an acetamide rather than an amine, thereby terminating polymerization.
[0044] Figure 24 depicts cleavage of the polycarbamate from the nucleotide backbone.
Desilylation of the enol ether linker attaching the anti-codon moiety to the monomer unit and the elimination of phosphate driven by the resulting release of phenol provides the provides the polycarbamate covalently linked at its carboxy terminus to its encoding single-stranded DNA.
[0045] Figure 25 depicts components of an amplifiable, evolvable furictionalized peptide nucleic acid library.
[0046] Figure 26 depicts test reagents used to optimize reagents and conditions for DNAtemplated PNA coupling.
[0047] Figure 27 depicts a simple set of PNA monomers derived from commercially available building blocks useful for evolving a PNA-based fluorescent Ni2+ sensor.
of 88 00
O
S[0048] Figure 28 depicts two schemes for the selection of a biotin-terminated functionalized ct PNA capable of catalyzing an aldol or retroaldol reaction.
cr 1 [0049] Figure 29 depicts DNA-template-directed synthesis of a combinatorial small
O
molecule library.
[0050] Figure 30 shows schematically how DNA-linked small molecule scaffolds can be functionalized sequence-specifically by reaction with synthetic reagents linked to complementary nucleic acid oligonucleotides, this process can be repeated to complete the 00 synthetic transformations leading to a fully functionalized molecule.
S[0051] Figure 31 shows the functionalization of a cephalosporin small molecule scaffold with various reactants.
[0052] Figure 32 depicts a way of measuring the rate of reaction between a fixed nucleophile and an electrophile hybridized at varying distances along a nucleic acid template to define an essential reaction window in which nucleic acid-templated synthesis of nonpolymeric structures can take place.
[0053] Figure 33A depicts three linker strategies for DNA-templated synthesis. In the autocleaving linker strategy, the bond connecting the product from the reagent oligonucleotide is cleaved as a natural consequence of the reaction. In the scarless and useful scar linker strategies, this bond is cleaved following the DNA-templated reaction. The depicted reactions were analyzed by denaturing polyacrylamide gel electrophoresis shown in Figure 33B. Lanes 1-3 were visualized using UV light without DNA staining; lanes 4-10 were visualized by staining with ethidium bromide following by UV transillumination. Conditions: 1 to 3: one equivalent each of reagent and template, 0.1 M TAPS buffer pH 8.5, 1 M NaC1, 25 1.5 h; 4 to 6: three equivalents of 4, 0.1 M MES buffer pH 7.0, 1 M NaNO 2 10 mM AgNO 3 37 8 h; 8 to 9: 0.1 M CAPS buffer pH 11.8, 60 mM BME, 37 2 h; 11 to 12: 50 mM aqueous NaIO 4 25 2 h.
R1 NH(CH 2 2 NH-dansyl; R 2 biotin.
[0054] Figure 34 depicts strategies for purifying products of DNA-templated synthesis.
Using biotinylated reagent oligonucleotides, products arising from an autocleaving linker are partially purified by washing the crude reaction with avidin-linked beads (top). Products generated from DNA-templated reactions using the scarless or useful scar linkers can be purified by using biotinylated reagent oligonucleotides, capturing crude reaction products with avidinlinked beads, and eluting desired products by inducing linker cleavage (bottom).
11 of 88 00
O
[0055] Figure 35 depicts the generation of an initial template pool for an exemplary library c synthesis.
C [0056] Figure 36 depicts the DNA-templated synthesis of a non-natural peptide library.
[0057] Figure 37 depicts a 5'-reagent DNA-linker-amino acid.
[0058] Figure 38 depicts the DNA-templated synthesis of an evolvable diversity oriented bicyclic library.
[0059] Figures 39A and 39B depict DNA-templated multi-step tripeptide synthesis. Each 0 DNA-templated amide formation used reagents containing the sulfone linker described in the text. Conditions: step 1: activate two equivalents 13 in 20 mM EDC, 15 mM sulfo-NHS, 0.1 M MES buffer pH 5.5, 1 M NaCI, 10 min, 25 then add to template in 0.1 M MOPS pH 7.5, 1M NaC1, 25 0 C, 1 h; steps 2 and 3: two equivalents of reagent, 50 mM DMT-MM, 0.1 M MOPS buffer pH 7.0, 1 M NaCI, 6 h, 25 Desired product after each step was purified by capturing on avidin-linked beads and eluting with 0.1 M CAPS buffer pH 11.8, 60 mM BME, 37 2 h.
The progress of each reaction and purification was followed by denaturing polyacrylamide gel electrophoresis (Fig. 39B). Lanes 3, 6, and 9: control reactions using reagents containing scrambled oligonucleotide sequences.
[0060] Figures 40A and 40B depict Non-peptidic DNA-templated multi-step synthesis. The reagent linkers used in steps 1, 2, and 3 were the diol linker, autocleaving Wittig linker, and sulfone linker, respectively; see Figure 1 for linker cleavage conditions. Conditions: 17 to 18: activate two equivalents 17 in 20 mM EDC, 15 mM sulfo-NHS, 0.1 M MES buffer pH 5.5, 1 M NaC1, 10 min, 25 then add to template in 0.1 M MOPS pH 7.5, 1M NaC1, 16°C, 8 h; 19 to 21: three equivalents 20, 0.1 M TAPS pH 9.0, 3 M NaCI, 48 h, 25 22 to 23: three equivalents 22, 0.1 M TAPS pH 8.5, 1 M NaC1, 21 h, 25 0 C. The progress of each reaction and purification was followed by denaturing polyacrylamide gel electrophoresis (Fig. 40B). Lanes 3, 6, and 9: control reactions using reagents containing scrambled oligonucleotide sequences.
[00611 Figure 41 depicts the use of nucleic acids to direct the synthesis of new polymers and plastics by attaching the nucleic acid to the ligand of a polymerization catalyst. The nucleic acid can fold into a complex structure which can affect the selectivity and activity of the catalyst.
[0062] Figure 42 depicts the use of Grubbs' ring-opening metathesis polymerization catalysis in evolving plastics. The synthetic scheme of a dihydroimidazole ligand attached to DNA is shown as well as the monomer to be used in the polymerization reaction.
12 of 88 00 C [0063] Figure 43 depicts the evolution of plastics through iterative cycles of ligand ct diversification, selection and amplification to create polymers with desired properties.
S [0064] Figure 44 depicts an exemplary scheme for the synthesis, in vitro selection and amplification of a library of compounds.
[0065] Figure 45 depicts exemplary templates for use in recombination.
S [0066] Figure 46 depicts several exemplary deoxyribunucleotides and ribonucleotides bearing modifications to groups that do not participate in Watson-Crick hydrogen bonding and are known to be inserted with high sequence fidelity opposite natural DNA templates.
00 [0067] Figure 47 depicts exemplary metal binding uridine and 7-deazaadenosine analogs.
S [0068] Figure 48 depicts the synthesis of analog [0069] Figure 49 depicts the synthesis of analog [0070] Figure 50 depicts the synthesis of 8-modified deoxyadenosine triphosphates.
[0071] Figure 51 depicts the results of an assay evaluating the acceptance of modified nuceotides by DNA polymerases.
[0072] Figure 52 depicts the synthesis of 7-deazaadenosine derivatives.
[0073] Figure 53 depicts certain exemplary nucleotide triphosphates.
[0074] Figure 54 depicts a general method for the generation of libraries of metal-binding polymers.
[0075] Figures 55 and 56 depict exemplary schemes for the in vitro selections for nonnatural polymer catalysts.
[0076] Figures 57A-57C depict an exemplary scheme for the in vitro selection of catalysts for Heck reactions (Figure 57A), hetero Diels-Alder reactions (Figure 57B) and aldol additions (Figure 57C).
DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION [0077] As discussed above, it would be desirable to be able to evolve and amplify chemical compounds including, but not limited to small molecules and polymers, in the same way that biopolymers such as polynucleotides and proteins can be amplified and evolved. It has been demonstrated that DNA-templated synthesis provides a possible means of translating the information in a sequence of DNA into a synthetic small molecule. In general, DNA templates linked to one reactant may be able to recruit a second reactive group linked to a complementary DNA molecule to yield a product. Since DNA hybridization is sequence-specific, the result of a 13 of 88 00
O
C DNA-templated reaction is the translation of a specific DNA sequence into a corresponding ct reaction product. As shown in Figures 2A and 2B, the ability of single-stranded nucleic acid r templates to catalyze the sequence-specific oligomerization of complementary oligonucleotides Inoue et al. J. Am. Chem. Soc. 1981, 103, 7666; T. Inou et al. J. Mol. Biol. 1984, 178, 669- 76) has been demonstrated. This discovery was soon followed by findings that DNA or RNA r templates could catalyze the oligomerization of complementary DNA or RNA mono-, di-, tri-, or 0 oligonucleotides Inoue et al. J Am. Chem. Soc. 1981, 103, 7666; L. E. Orgel et al. Ace.
Chem. Res. 1995, 28, 109-118; H. Rembold et al. J. Mol. Evol. 1994, 38, 205; L. Rodriguez et al.
00 S J. Mol. Evol. 1991, 33, 477; C. B. Chen et al. J. Mol. Biol. 1985, 181, 271). DNA or RNA C1 templates have since been shown to accelerate the formation of a variety of non-natural nucleic acid analogs, including peptide nucleic acids Bohler et al. Nature 1995, 376, 578), phosphorothioate- K. Herrlein et al. J. Am. Chem. Soc. 1995, 117, 10151-10152), phosphoroselenate- Xu et al. J. Am. Chem. Soc. 2000, 122, 9040-9041; Y. Xu et al. Nat.
Biotechnol. 2001, 19, 148-152) and phosphoramidate-(A. Luther et al. Nature 1998, 396, 245-8) containing nucleic acids, non-ribose nucleic acids Bolli et al. Chem. Biol. 1997, 4, 309-20), and DNA analogs in which a phosphate linkage has been replaced with an aminoethyl group (Y.
Gat et al. Biopolymers 1998, 48, 19-28). Nucleic acid templates can also catalyze amine acylation between nucleotide analogs K. Bruick et al. Chem. Biol. 1996, 3, 49-56).
[0078] However, although the ability of nucleic acid templates to accelerate the formation of a variety of non-natural nucleic acid analogues has been demonstrated, nearly all of these reactions previously shown to be catalyzed by nucleic acid templates were designed to proceed through transition states closely resembling the structure of the natural nucleic acid backbone (Figs. 2A and 2B), typically affording products that preserve the same six-bond backbone spacing between nucleotide units. The motivation behind this design was presumably the assumption that the rate enhancement provided by nucleic acid templates depends on a precise alignment of reactive groups, and the precision of this alignment is maximized when the reactants and products mimic the structure of the DNA and RNA backbones. Evidence in support of the hypothesis that DNA-templated synthesis can only generate products that resemble the nucleic acid backbone comes from the well-known difficulty of macrocyclization in organic synthesis Illuminati et al. Ace. Chem. Res. 1981, 14, 95-102; R. B. Woodward et al. J. Am.
Chem. Soc. 1981, 103, 3210-3213). The rate enhancement of intramolecular ring closing reactions compared with their 14 of 88 nW7fnfta n/'Tv/T f l/io A ,ic 00YJ UVIUI197 Y ILI .)UUOJ9u 0 O intermolecular counterparts is known to diminish quickly as rotatable bonds are added between Sreactive groups, such that linking reactants with a flexible 14-carbon linker hardly affords any C. rate acceleration Illuminati et al. Acc. Chem. Res. 1981, 14, 95-102).
M
r [0079] Because synthetic molecules of interest do not in general resemble nucleic acid backbones, the use of DNA-templated synthesis to translate DNA sequences into synthetic small molecules would be broadly useful only if synthetic molecules other than nucleic acids and Snucleic acid analogs could be synthesized in a DNA-templated fashion. The ability of DNA- Stemplated synthesis to translate DNA sequences into arbitrary non-natural small molecules C therefore requires demonstrating that DNA-templated synthesis is a much more general 00 phenomenon than has been previously described.
C [0080] Signficantly, for the first time it has been demonstrated herein that DNA-templated synthesis is indeed a general phenomenon and can be used for a variety of reactions and conditions to generate a diverse range of compounds, specifically including for the first time, compounds that are not, or do not resemble, nucleic acids or analogs thereof. More specifically, the present invention extends the ability to amplify and evolve libraries of chemical compounds beyond natural biopolymers. The ability to synthesize chemical compounds of arbitrary structure allows researchers to write their own genetic codes incorporating a wide range of chemical functionality into novel backbone and side-chain structures, which enables the development of novel catalysts, drugs, and polymers, to name a few examples. For example, the ability to directly amplify and evolve these molecules by genetic selection enables the discovery of entirely new families of artificial catalysts which possess activity, bioavailability, solvent, or thermal stability, or other physical properties (such as fluorescence, spin-labeling, or photolability) which are difficult or impossible to achieve using the limited set of natural protein and nucleic acid building blocks. Similarly, developing methods to amplify and directly evolve synthetic small molecules by iterated cycles of mutation and selection enables the isolation of novel ligands or drugs with properties superior to those isolated by traditional rational design or combinatorial screening drug discovery methods. Additionally, extending the approaches described herein to polymers of significance in material science would enable the evolution of new plastics.
[0081] In general, the method of the invention involves 1) providing one or more nucleic acid templates, which one or more nucleic acid templates optionally have a reactive unit of 88 wVn n f AQ7 r3rfqPrITr I/nQCAK associated therewith; and 2) contacting the one or more nucleic acid templates with one or more Stransfer units designed to have a first moiety, an anti-codon which hybridizes to a sequence of -s the nucleic acid, andis associated with a second moiety, a reactive unit, which includes specific functionality, a building block, reactant, etc. for the compound to be synthesized. It will be appreciated that in certain embodiments of the invention, the transfer unit comprises one moiety incorporating the hybridization capablility of the anti-codon unit and the chemical functionality of the reaction unit. Once these transfer units have hybridized to the nucleic acid template in a sequence-specific manner, the synthesis of the chemical compound can take place due to the interaction of reactive units present on the transfer units and/or the nucleic acid template.
00 Significantly, the sequence of the nucleic acid can later be determined to decode the synthetic C,1 history of the attached compound and thereby its structure. It will be appreciated that the method described herein may be used to synthesize one molecule at a time or may be iused to synthesize thousands to millions of compounds using combinatorial methods.
[0082] It will be appreciated that a variety of chemical compounds can be prepared and evolved according to the method of the invention. In certain embodiments of the invention, however, the methods are utilized for the synthesis of chemical compounds that are not, or do not, resemble nucleic acids or nucleic acid analogs. For example, in certain embodiments of the invention, small molecule compounds can be syntheiszed by providing a template which has a reactive unit building block or small molecule scaffold) associated therewith (attached directly or through a linker as described in more detail in Examples 5 herein), and contacting the template simultaneously or sequentially with one or more transfer units having one or more reactive units associated therewith. In certain other embodiments, non-natural polymers can be synthesized by providing a template and contacting the template simultaneously with one or more transfer units having one or more reactive units associated therewith under conditions suitable to effect reaction of the adjacent reactive units on each of the transfer units (see, for example, Figure 3, and examples 5 and 9, as described in more detail herein).
[0083] Certain embodiments are discussed in more detail below; however, it will be appreciated that the present invention is not intended to be limited to those embodiments discussed below. Rather, the present invention is intended to encompass these embodiments and equivalents thereof.
16 of 88 ~-m~in rn PCrTis042/n8546 00 WU 0IU 14YLY Templates S[0084] As discussed above, one or more templates are utilized in the method of the invention and hybridize to the transfer units to direct the synthesis of the chemical compound. As would S be appreciated by one of skill in this art, any template may be used in the methods and compositions of the present invention. Templates which can be mutated and thereby evolved can be used to guide the synthesis of another chemical compound or library of chemical compounds Sas described in the present invention. As described in more detail herein, the evolvable template encodes the synthesis of a chemical compound and can be used later to decode the synthetic 00 history of the chemical compound, to indirectly amplify the chemical compound, and/or to Sevolve diversify, select, and amplify) the chemical compound. The evolvable template is,
C
1 in certain embodiments, a nucleic acid. In certain embodiment of the present invention, the template is based on a nucleic acid.
[0085] The nucleic acid templates used in the present invention are made of DNA, RNA, a hybrid of DNA and RNA, or a derivative of DNA and RNA, and may be single- or doublestranded. The sequence of the template is used in the inventive method to encode the synthesis of a chemical compound, preferably a compound that is not, or does not resemble, a nucleic acid or nucleic acid analog an unnatural polymer or a small molecule). In the case of certain unnatural polymers, the nucleic acid template is used to align the monomer units in the sequence they will appear in the polymer and to bring them in close proximity with adjacent monomer units along the template so that they will react and become joined by a covalent bond. In the case of a small molecule, the template is used to bring particular reactants within proximity of the small molecule scaffold in order that they may modify the'scaffold in a particular way. In certain other embodiments, the template can be utilized to generate non-natural polymers by PCR amplification of a synthetic DNA template library consisting of a random region of nucleotides, as describe in Example 9 herein.
[0086] As would be appreciated by one of skill in the art, the sequence of the template may be designed in a number of ways without going beyond the scope of the present invention. For example, the length of the codon must be determined and the codon sequences must be set. If a codon length of two is used, then using the four naturally occurring bases only 16 possible combinations are available to be used in encoding the library. If the length of the codon is increased to three (the number Nature uses in encoding proteins), the number of possible 17 of 88 wn fn/n7499 PC1T/US02/08546 00 combinations is increased to 64. Other factors to be considered in determining the length of the Scodon are mismatching, frame-shifting, complexity of library, etc. As the length of the codon is .s increased up to a certain extent the number of mismatches is decreased; however, excessively long codons will hybridize despite mismatched base pairs. In certain embodiments of special interest, the length of the codon ranges between 2 and 10 bases.
i.t [0087] Another problem associated with using a nucleic acid template is frame shifting. In Nature, the problem of frame-shifting in the translation of protein from an mRNA is avoided by Suse of the complex machinery of the ribosome. The inventive methods, however, will not take Sadvantage of such complex machinery. Instead, frameshifting may be remedied by lengthening 00 each codon such that hybridization of a codon out of frame will guarantee a mismatch. For C,1 example, each codon may start with a G, and subsequent positions may be restricted to T, C, and A (Figure In another example, each codon may begin and end with a G, and subsequent positions may be restricted to T, C, and A. Another way of avoiding frame shifting is to have the codons sufficiently long so that the sequence of the codon is only found within the sequence of the template "in frame". Spacer sequences may also be placed in between the codons to prevent frame shifting.
[0088] It will be appreciated that the template can vary greatly in the number of bases. For example, in certain embodiments, the template may be 10 to 10,000 bases long, preferably between 10 and 1,000 bases long. The length of the template will of course depend on the length of the codons, complexity of the library, length of the unnatural polymer to be synthesized, complexity of the small molecule to be synthesized, use of space sequences, etc. The nucleic acid sequence may be prepared using any method known in the art to prepare nucleic acid sequences. These methods include both in vivo and in vitro methods including PCR, plasmid preparation, endonuclease digestion, solid phase synthesis, in vitro transcription, strand separation, etc. In certain embodiments, the nucleic acid template is synthesized using an automated DNA synthesizer.
[0089] As discussed above, in certain embodiments of the invention, the method is used to synthesize chemical compounds that are not, or do not resemble, nucleic acids or nucleic acid analogs. Although it has been demonstrated that DNA-templated synthesis can be utilized to direct the synthesis of nucleic acids and analogs thereof, it has not been previously demonstrated that the phenomenon of DNA-tempalted synthesis is general enough to extend to other more 18 of 88 WO 02/074929 PCT/US02/08546 00 0 O complex chemical compounds small molecules, non-natural polymers). As described in Sdetail herein, it has been demonstrated that DNA-templated synthesis is indeed a more general Sphenomenon and that a variety of reactions can be utilized.
c [0090] Thus, in certain embodiments of the present invention, the nucleic acid template comprises sequences of bases that encode the synthesis of an unnatural polymer or small t" molecule. The message encoded in the nucleic acid template preferably begins with a specific C, codon that bring into place a chemically reactive site from which the polymerization can take Splace, or in the case of synthesizing a small molecule the "start" codon may encode for an anti- C1 codon associated with a small molecule scaffold or a first reactant. The "start" codon of the 00 present invention is analogous to the "start" codon, ATG, found in Nature, which encodes for the amino acid methionine. To give but one example for use in synthesizing an unnatural polymer library, the start codon may encode for a start monomer unit comprising a primary amine masked by a photolabile protecting group, as shown below in Example [0091] In yet other embodiments of the invention, the nucleic acid template itself may be modified to include an initiation site for polymer synthesis a nucleophile) or a small molecule scaffold. In certain embodiments, the nucleic acid template includes a hairpin loop on one of its ends terminating in a reactive group used to initiate polymerization of the monomer units. For example, a DNA template may comprise a hairpin loop terminating in a group, which may be protected or not. From the amino group polymerization of the unnatural polymer may commence. The reactive amino group can also be used to link a small molecule scaffold onto the nucleic acid template in order to synthesize a small molecule library.
[0092] To terminate the synthesis of the unnatural polymer a "stop" codon should be included in the nucleic acid template preferably at the end of the encoding sequence. The "stop" codon of the present invention is analogous to the "stop" codons TAA, TAG, TGA) found in mRNA transcripts. In Nature, these codons lead to the termination of protein synthesis. In certain embodiments, a "stop" codon is chosen that is compatible with the artificial genetic code used to encode the unnatural polymer. For example, the "stop" codon should not conflict with any other codons used to encode the synthesis, and it should be of the same general format as the other codons used in the template. The "stop" codon may encode for a monomer unit that terminates polymerization by not providing a reactive group for further attachment. For example, a stop monomer unit may contain a blocked reactive group such as an acetamide rather 19 of 88 WO 02/074929 PCT/US02/08546 00
O
O than a primary amine as shown in Example 5A below. In yet other embodiments, the stop Smonomer unit comprises a biotinylated terminus providing a convenient way of terminating the c. polymerization step and purifying the resulting polymer.
Transfer Units [0093] As described above, in the method of the invention, transfer units are also provided which comprise an anti-codon and a reactive unit. It will be appreciated that the anti-codons O used in the present invention are designed to be complementary to the codons present within the C"1 nucleic acid template, and should be designed with the nucleic acid template and the codons used 00 Stherein in mind. For example, the sequences used in the template as well as the length of the codons would need to be taken into account in designing the anti-codons. Any molecule which is complementary to a codon used in the template may be used in the inventive methods nucleotides or non-natural nucleotides). In certain embodiments, the codons comprise one or more bases found in nature thymidine, uracil, guanidine, cytosine, adenine). In certain other embodiments, the anti-codon comprises one or more nucleotides normally found in Nature with a base, a sugar, and an optional phosphate group. In yet other embodiments, the bases are strung out along a backbone that is not the sugar-phosphate backbone normally found in Nature non-natural nucleotides).
[0094] As discussed above, the anti-codon is associated with a particular type of reactive unit to form a transfer unit. It will be appreciated that this reactive unit may represent a distinct entity or may be part of the functionality of the anti-codon unit (see, Example In certain embodiments, each anti-codon sequence is associated with one monomer type. For example, the anti-codon sequence ATTAG may be associated with a carbamate residue with an iso-butyl side chain, and the anti-codon sequence CATAG may be associated with a carbamate residue with a phenyl side chain. This one-for-one mapping of anti-codon to monomer units allows one to decode any polymer of the library by sequencing the nucleic acid template used in the synthesis and allows one to synthesize the same polymer or a related polymer by knowing the sequence of the original polymer. It will be appreciated by one of skill in this art that by changing mutating) the sequence of the template, different monomer units will be brought into place, thereby allowing the synthesis of related polymers, which can subsequently be selected and of 88 WO 02/074929 PrT/fT /Oni/f 46 00 0 evolved. In certain preferred embodiments, several anti-codons may code for one monomer unit Sas is the case in Nature.
[0095] In certain other embodiments of the present invention where a small molecule library r is to be created rather than a polymer library, the anti-codon is associated with a reactant used to modify the small molecule scaffold. In certain embodiments, the reactant is associated with the anti-codon through a linker long enough to allow the reactant to come in contact with the small S molecule scaffold. The linker should preferably be of such a length and composition to allow for intramolecular reactions and minimize intermolecular reactions. The reactants include a variety N of reagents as demonstrated by the wide range of reactions that can be utilized in DNA- Stemplated synthesis (see Example 2, 3 and 4 herein) and can be any chemical group, catalyst N organometallic compounds), or reactive moiety electrophiles, nucleophiles) known in the chemical arts.
[0096] Additionally, the association between the anti-codon and the monomer unit or reactant in the transfer unit may be covalent or non-covalent. In certain embodiments of special intereste, the association is through a covalent bond, and in certain embodiments the covalent linkage is severable. The linkage may be cleaved by light, oxidation, hydrolysis, exposure to acid, exposure to base, reduction, etc. For examples of linkages used in this art, please see Fruchtel et al. Angew. Chem. Int. Ed. Engl. 35:17, 1996, incorporated herein by reference. The anti-codon and the monomer unit or reactant may also be associated through non-covalent interactions such as ionic, electrostatic, hydrogen bonding, van der Waals interactions, hydrophobic interactions, pi-stacking, etc. and combinations thereof To give but one example, the anti-codon may be linked to biotin, and the monomer unit linked to streptavidin. The propensity of streptavidin to bind biotin leads to the non-covalent association between the anticodon and the monomer unit to form the transfer unit.
Synthesis of Certain Exemplary Compounds [0097] It will be appreciated that a variety of compounds and/or libraries can be prepared using the method of the invention. As discussed above, in certain embodiments of special interest, compounds that are not, or do not resemble, nucleic acids or analogs thereof, are synthesized according to the method of the invention.
21 of 88 WO 02/074929 PrT/UTSn7/n546 00 O [0098] In certain embodiments, polymers, specifically unnatural polymers, are prepared Saccording to the method of the present invention. The unnatural polymers that can be created using the inventive method and system include any unnatural polymers. Exemplary unnatural r^ polymers include, but are not limited to, polycarbamates, polyureas, polyesters, polyacrylate, polyalkylene polyethylene, polypropylene), polycarbonates, polypeptides with unnatural Sstereochemistry,.polypeptides with unnatural amino acids, and combination thereof. In certain 1 embodiments, the polymers comprises at least 10 monomer units. In certain other embodiments, Sthe polymers comprise at least 50 monomer units. In yet other embodiments, the polymers CN comprise at least 100 monomer units. The polymers synthesized using the inventive system may 00 0be used as catalysts, pharmaceuticals, metal chelators, materials, etc.
[0099] In preparing certain unnatural polymers, the monomer units attached to the anticodons and used in the present invention may be any monomers or oligomers capable of being joined together to form a polymer. The monomer units may be carbamates, D-amino acids, unnatural amino acids, ureas, hydroxy acids, esters, carbonates, acrylates, ethers, etc. In certain embodiments, the monomer units have two reactive groups used to link the monomer unit into the growing polymer chain. Preferably, the two reactive groups are not the same so that the monomer unit may be incorporated into the polymer in a directional sense, for example, at one end may be an electrophile and at the other end a nucleophile. Reactive groups may include, but are not limited to, esters, amides, carboxylic acids, activated carbonyl groups, acid chlorides, amines, hydroxyl groups, thiols, etc. In certain embodiments, the reactive groups are masked or protected (Greene Wuts Protective Groups in Organic Synthesis, 3rd Edition Wiley, 1999; incorporated herein by reference) so that polymerization may not take place until a desired time when the reactive groups are deprotected. Once the monomer units are assembled along the nucleic acid template, initiation of the polymerization sequence results in a cascade of polymerization and deprotection steps wherein the polymerization step results in deprotection of a reactive group to be used in the subsequent polymerization step (see, Figure 3).
[00100] The monomer units to be polymerized may comprise two or more units depending on the geometry along the nucleic acid template. As would be appreciated by one of skill in this art, the monomer units to be polymerized must be able to stretch along the nucleic acid template and particularly across the distance spanned by its encoding anti-codon and optional spacer sequence.
In certain embodiments, the monomer unit actually comprises two monomers, for example, a 22 of 88 WO 02/074929 PCT/US02/08546 00 dicarbamate, a diurea, a dipeptide, etc. In yet other embodiments, the monomer unit actually comprises three or more monomers.
S[00101] The monomer units may contain any chemical groups known in the art. As would be C< appreciated by one of skill in this art, reactive chemical groups especially those that would interfere with polymerization, hybridization, etc. are masked using known protecting groups (Greene Wuts Protective Groups in Organic Synthesis, 3rd Edition Wiley, 1999; incorporated C,1 herein by reference). In general, the protecting groups used to mask these reactive groups are orthogonal to those used in protecting the groups used in the polymerization steps.
[00102] In synthesizing an unnatural polymer, in certain embodiments, a template is provided 00 Sencoding the sequence of monomer units. Transfer units are then allow to contact the template Sunder conditions that allow for hybridization of the anti-codons to the template. Polymerization of the monomer units along the template is then allowed to occur to form the unnatural polymer.
The newly synthesized polymer may then be cleaved from the anti-codons and/or the template.
The template may be used as a tag to elucidate the structure of the polymer or may be used to amplify and evolve the unnatural polymer. As will be described in more detail below, the present method may be used to prepare a library of unnatural polymers. For example, in certain embodiments, as described in more detail in Example 9 herein, a library of DNA templates can be utilized to prepare unnatural polymers. In general, the method takes advantage of the fact that certain DNA polymerases are able to accept certain modified nucleotide triphosphate substrates and that several deoxyribonucleotides and ribonucleotides bearing modified groups that do not participate in Watson-Crick bonding are known to be inserted with high sequence specificity opposite natural DNA templates. Accordingly, single stranded DNA containing modified nucleotides can serve as efficient templates for the DNA-polymerase catalyzed incorporation of natural or modified nucleotides.
[00103] It will be appreciated that the inventive methods may also be used to synthesize other classes of chemical compounds besides unnatural polymers. For example, small molecules may be prepared using the methods and compositions provided by the present invention. These small molecules may be natural product-like, non-polymeric, and/or non-oligomeric. The substantial interest in small molecules is due in part to their use as the active ingredient in many pharmaceutical preparations although they may also be used as catalysts, materials, additives, etc.
23 of 88 WO 02/074929 PCT/US02/08546 00 O [00104] In synthesizing small molecules using the method of the present invention, an evolvable template is also provided. The template may either comprise a small molecule c scaffold upon which the small molecule is to be built, or a small molecule scaffold may be added cr to the template. The small molecule scaffold may be any chemical compound with sites for functionalization. For example, the small molecule scaffold'may comprises a ring system the ABCD steroid ring system found in cholesterol) with functionalizable groups off the atoms C1 making up the rings. In another example, the small molecule may be the underlying structure of 0a pharmaceutical agent such as morphine or a cephalosporin antibiotic (see Examples 5C and below below). The sites or groups to be functionalized on the small molecule scaffold may be 0 protected using methods and protecting groups known in the art. The protecting groups used in a Ssmall molecule scaffold may be orthogonal to one another so that protecting groups can be removed one at a time.
[00105] In this embodiment, the transfer units comprise an anti-codon similar to those described in the unnatural polymer synthesis; however, these anti-codons are associated with reactants or building blocks to be used in modifying, adding to, or taking away from the small molecule scaffold. The reactants or building blocks may be electrophiles acetyl, amides, acid chlorides, esters, nitriles, imines), nucleophiles amines, hydroxyl groups, thiols), catalysts organometallic catalysts), side chains, etc. See, for example reactions in aqueous and organic media as described herein in Examples 2 and 4. The transfer units are allowed to contact the template under hydridizing conditions, and the attached reactant or building block is allowed to react with a site on the small molecule scaffold. In certain embodiments, protecting groups on the small molecule template are removed one at a time from the sites to be functionalized so that the reactant of the transfer unit will react at only the desired position on the scaffold. As will be appreciated by one of skill in the art, the anti-codon may be associated with the reactant through a linker moiety (see, Example The linker facilitates contact of the reactant with the small molecule scaffold and in certain embodiments, depending on the desired reaction, positions DNA as a leaving group ("autocleavable" strategy), or may link reactive groups to the template via the "scarless" linker strategy (which yields product without leaving behind additional chemical functionality), or a "useful scar" strategy (in which the linker is left behind and can be functionalized in subsequent steps following linker cleavage). The reaction condition, linker, reactant, and site to be functionalized are chosen to avoid intermolecular 24 of 88 WO 02/074929 PCT/US02/08546 00 reactions and accelerate intramolecular reactions. It will also be appreciated that the method of "-the present invention contemplates both sequential and simultaneous contacting of the template with transfer units depending on the particular compound to be synthesized. In certain Mc embodiments of special interest, the multi-step synthesis of chemical compounds is provided in which the template is contacted sequentially with two or more transfer units to facilitate multistep synthesis of complex chemical compounds.
[001061 After the sites on the scaffold have been modified, the newly synthesized small molecule is linked to the template that encoded is synthesis. Decoding of the template tag will C allow one to elucidate the synthetic history and thereby the structure of the small molecule. The 00 template may also be amplified in order to create more of the desired small molecule and/or the c, template may be evolved to create related small molecules. The small molecule may also be cleaved from the template for purification or screening.
[00107] As would be appreciated by one of skill in this art, a plurality of templates may be used to encode the synthesis of a combinatorial library of small molecules using the method described above. This would allow for the amplification and evolution of a small molecule library, a feat which has not been accomplished before the present invention.
Method ofSynthesizing Libraries of Compounds [00108] In the inventive method, a nucleic acid template, as described above, is provided to direct the synthesis of an unnatural polymer, a small molecule, or any other type of molecule of interest. In general, a plurality of nucleic acid templates is provided wherein the number of different sequences provided ranges from 2 to 101 5 In one embodiment of the present invention, a plurality of nucleic acid templates is provided, preferably at least 100 different nucleic acid templates, more preferably at least 10000 different nucleic acid templates, and most preferably at least 1000000 different nucleic acid templates. Each template provided comprises a unique nucleic acid sequence used to encode the synthesis of a particular unnatural polymer or small molecule. As described above, the template may also have functionality such as a primary amine from which the polymerization is initiated or a small molecule scaffold. In certain embodiments, the nucleic acid templates are provided in one "pot". In certain other embodiments, the templates are provided in aqueous media, and subsequent reactions are performed in aqueous media.
of 88 WO 02/074929 PCT/US02/08546 00
O
O [00109] To the template is added transfer units with anti-codons, as described above, associated with a monomer unit, as described above. In certain embodiments, a plurality of transfer units is provided so that there is an anti-codon for every codon represented in the CM template. In a preferred embodiment, certain anti-codons are used as start and stop sites. In general, a large enough number of transfer units is provided so that all corresponding codon sites on the template are filled after hybridization.
CK [00110] The anti-codons of the transfer units are allowed to hybridize to the nucleic acid Stemplate thereby bringing the monomer units together in a specific sequence as determined by Cf the template. In the situation where a small molecule library is being synthesized, reactants are 00 Sbrought in proximity to a small molecule scaffold. The hybridization conditions, as would be appreciated by those of skill in the art, should preferably allow for only perfect matching between the codon and its anti-codon. Even single base pair mismatches should be avoided.
Hybridization conditions may include, but are not limited to, temperature, salt concentration, pH, concentration of template, concentration of anti-codons, and solvent. The hybridization conditions used in synthesizing the library may depend on the length of the codon/anti-codon, the similarity between the codons present in the templates, the content of G/C versus A/T base pairs, etc (for further information regarding hybridization conditions, please see, Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Nucleic Acid Hybridization D. Hames S. J. Higgins eds.
1984); the treatise, Methods in Enzymology (Academic Press, Inc., Ausubel et al. Current Protocols in Molecular Biology (John Wiley Sons, Inc., New York, 1999); each of which is incorporated herein by reference).
[00111] After hybridization of the anti-codons to the codons on the template have occurred, the monomer units are then polymerized in the case of the synthesis of unnatural polymers. The polymerization of the monomer units may occur spontaneously or may need to be initiated, for example, by the deprotection of a reactive groups such as a nucleophile or by providing light of a certain wavelength. In certain other embodiments, polymers can be catalyzed by DNA polymerization capable of effecting polymerization of non-natural nucleotides (see, Example 9).
The polymerization preferably occurs in one direction along the template with adjacent monomer units becoming joined through a covalent linkage. The termination of the polymerization step occurs by the addition of a monomer unit that is not capable of being added onto. In the case of 26 of 88 WO 02/074929 PCT/US02/08546 00 the synthesis of small molecules, the reactants are allowed to react with the small molecule scaffold. The reactant may react spontaneously, or protecting groups on the reactant and/or the small molecule scaffold may need to be removed. Other reagents acid, base, catalyst, Mc, hydrogen gas, etc.) may also be needed to effect the reaction (see, Examples [00112] After the unnatural polymers or small molecules have been created with the aid of the nucleic acid template, they may be cleaved from the nucleic acid template and/or anti-codons (1 used to synthesize them. In certain embodiments, the polymers or small molecules are assayed before being completely detached from the nucleic acid templates that encode them. Once the (1 polymer or small molecule is selected, the sequence of the template or its complement may be 00 0determined to elucidate the structure of the attached polymer or small molecule. This sequence may then be amplified and/or evolved to create new libraries of related polymers or small molecules that in turn may be screened and evolved.
Uses [00113] The methods and compositions of the present invention represent a new way to generate molecules with desired properties. This approach marries the extremely powerful genetic methods, which molecular biologists have taken advantage of for decades, with the flexibility and power of organic chemistry. The ability to prepare, amplify, and evolve unnatural polymers by genetic selection may lead to new classes of catalysts that possess activity, bioavailability, stability, fluorescence, photolability, or other properties that are difficult or impossible to achieve using the limited set of building blocks found in proteins and nucleic acids.
Similarly, developing new systems for preparing, amplifying, and evolving small molecules by iterated cycles of mutation and selection may lead to the isolation of novel ligands or drugs with properties superior to those isolated by slower traditional drug discovery methods (see, Example 7).
100114] Performing organic library synthesis on the molecular biology scale is a fundamentally different approach from traditional solid phase library synthesis and carries significant advantages. A library created using the inventive methods can be screened using any method known in this art binding assay, catalytic assay). For example, selection based on binding to a target molecule can be carried out on the entire library by passing the library over a resin covalently linked to the target. Those biopolymers that have affinity for the resin-bound 27 of 88 WO 02/074929 PCT/US02/08546 00 O target can be eluted with free target molecules, and the selected compounds can be amplified using the methods described above. Subsequent rounds of selection and amplification can result c in a pool of compounds enriched with sequences that bind the target molecule. In certain (cf embodiments, the target molecule mimics a transition state of a chemical reaction, and the chemical compounds selected may serve as a catalyst for the chemical reaction. Because the information encoding the synthesis of each molecule is covalently attached to the molecule at 1 one end, an entire library can be screened at once and yet each molecule is selected on an 0individual basis.
c, [00115] Such a library can also be evolved by introducing mutations at the DNA level using 0 error-prone PCR (Cadwell et al. PCR Methods Appl. 2:28, 1992; incorporated herein by Sreference) or by subjecting the DNA to in vitro homologous recombination (Stemmer Proc. Natl.
Acad. Sci. USA 91:10747, 1994; Stemmer Nature 370:389, 1994; each of which is incorporated herein by reference). Repeated cycled of selection, amplification, and mutation may afford biopolymers with greatly increased binding affinity for target molecules or with significantly improved catalytic properties. The final pool of evolved biopolymers having the desired properties can be sequenced by sequencing the nucleic acid cleaved from the polymers. The nucleic acid-free polymers can be purified using any method known in the art including HPLC, column chromatography, FLPC, etc., and its binding or catalytic properties can be verified in the absence of covalently attached nucleic acid.
[00116] The polymerization of synthetically-generated monomer units independent of the ribosomal machinery allows the incorporation of an enormous variety of side chains with novel chemical, biophysical, or biological properties. Terminating each biopolymer with a biotin side chain, for example, allows the facile purification of only full-length biopolymers which have been completely translated by passing the library through an avidin-linked resin. Biotinterminated biopolymers can be selected for the actual catalysis of bond-breaking reactions by passing these biopolymers over resin linked through the substrate to avidin (Figure Those biopolymers that catalyze substrate cleavage would self-elute from a column charged with this resin. Similarly, biotin-terminated biopolymers can be selected for the catalysis of bond-forming reactions (Figure One substrate is linked to resin and the second substrate is linked to avidin.
Biopolymers that catalyze bond formation between the substrates are selected by their ability to ligate the substrates together, resulting in attachment of the biopolymer to the resin. Novel side 28 of 88 WO 02/074929 PCT/US02/08546 00
O
chains can also be used to introduce cofactor into the biopolymers. A side chain containing a metal chelator, for example, may provide biopolymers with metal-mediated catalytic properties, S while a flavin-containing side chain may equip biopolymers with the potential to catalyze redox Mc) reactions.
[00117] In this manner unnatural biopolymers may be isolated which serve as artificial receptors to selectively bind molecules or which catalyze chemical reactions. Characterization
C
of these molecules would provide important insight into the ability of polycarbamates, polyureas, polyesters, polycarbonates, polypeptides with unnatural side chain and stereochemistries, or other unnatural polymers to form secondary or tertiary structures with binding or catalytic 00 Sproperties.
Kits [00118] The present invention also provides kits and compositions for use in the inventive methods. The kits may contain any item or composition useful in practicing the present invention. The kits may include, but is not limited to, templates, anticodons, transfer units, monomer units, building blocks, reactants, small molecule scaffolds, buffers, solvents, enzymes heat stable polymerase, reverse transcriptase, ligase, restriction endonuclease, exonuclease, Klenow fragment, polymerase, alkaline phosphatase, polynucleotide kinase), linkers, protecting groups, polynucleotides, nucleosides, nucleotides, salts, acids, bases, solid supports, or any combinations thereof.
[001191 As would be appreciated by one of skill in this art, a kit for preparing unnatural polymers would contain items needed to prepare unnatural polymers using the inventive methods described herein. Such a kit may include templates, anti-codons, transfer units, monomers units, or combinations thereof. A kit for synthesizing small molecules may include templates, anticodons, transfer units, building blocks, small molecule scaffolds, or combinations thereof.
[00120] The inventive kit may also be equipped with items needed to amplify and/or evolve a polynucleotide template such as a heat stable polymerase for PCR, nucleotides, buffer, and primers. In certain other embodiments, the inventive kit includes items commonly used in performing DNA shuffling such as polynucleotides, ligase, and nucleotides.
29 of 88 WO 02/074929 PCT/US02/08546 00 0 O [00121] In addition to the templates and transfer units described herein, the present invention 'also includes compositions comprising complex small molecules, scaffolds, or unnatural polymer prepared by any one or more of the methods of the invention as described herein.
EQUIVALENTS
The representative examples that follow are intended to help illustrate the invention, and C are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, Svarious modifications of the invention and many further embodiments thereof, in addition to 00 C1 those shown and described herein, will become apparent to those skilled in the art from the full Scontents of this document, including the examples which follow and the references to the C, scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art.
The following examples contain important additional information, exemplification and guidance that can be adapted to the.practice of this invention in its various embodiments and the equivalents thereof.
EXEMPLIFICATION
[00122] Example 1: The Generality of DNA-Templated Synthesis: Clearly, implementing the small molecule evolution approach described above requires establishing the generality of DNA-templated synthesis. The present invention, for the first time, establishes the generality fo this approach and thus enables the syntheis of a vareity of chemical compounds using DNAtemplated synthesis. As shown in Figure 6a, the ability of two DNA architectures to support solution-phase DNA-templated synthesis was established. Both hairpin and end-of-helix (E) templates bearing electrophilic maleimide groups reacted efficiently with one equivalent of thiol reagent linked to a complementary DNA oligonucleotide to yield the thioether product in minutes at 25 DNA-templated reaction rates (kapp ~10 5 M-'s 1 were similar for H and E architectures despite significant differences in the relative orientation of their reactive groups. In contrast, no product was observed when using reagents containing sequence mismatches, or when using templates pre-quenched with excess P-mercaptoethanol (Fig. 6a). Both templates therefore support the sequence-specific DNA-templated addition of a thiol to a maleimide even though the structures of the resulting products differ markedly from the structure of the natural of 88 WO 02/074929 PCT/US02/08546 00 0 O DNA backbone. Little or no non-templated intermolecular reaction products are observed under Sthe reaction conditions (pH 7.5, 25 OC, 250 mM NaC1, 60 nM template and reagent).
[00123] Additionally, sequence-specific DNA-templated reactions spanning a variety of eC reaction types (SN2 substitutions, additions to a,p-unsaturated carbonyl systems, and additions to vinyl sulfones), nucleophiles (thiols and amines), and reactant structures all proceeded in good yields with excellent sequence selectivity (Fig. 6b). Expected product masses were verified by C1 mass spectrometry. In each case, matched but not mismatched reagents afforded product Sefficiently despite considerable variations in their transition state geometry, steric hindrance, and Sconformational flexibility. Collectively these findings indicate that DNA-templated synthesis is 00 Sa general phenomenon capable of supporting a range of reaction types, and is not limited to the 'i creation of structures resembling nucleic acid backbones as described previously.
[00124] Since sequence discrimination is important for the faithful translation of DNA into synthetic structures, the reaction rate of a matched reagent compared with that of a reagent bearing a single mismatched base near the center of its 10-base oligonucleotide was measured.
At 25. C, the initial rate of reaction of matched thiol reagents with iodoacetamide-linked H templates is 200-fold faster than that of reagents bearing a single mismatch (kpp 2.4 x 10 4 M-'s Svs. 1.1 x 102 MvTs'', Fig. In addition, small amounts of products arising from the annealing of mismatched reagents can be eliminated by elevating the reaction temperature beyond the T, of the mismatched reagents (Fig. The decrease in the rate of product formation as temperature is elevated further indicates that product formation proceeds by a DNA-templated mechanism rather than a simple intermolecular mechanism.
[001251 In addition to reaction generality and sequence specificity, DNA-templated synthesis also demonstrates remarkable distance independence. Both H and E templates linked to maleimide or ac-iodoacetamide groups promote sequence-specific reaction with matched, but not mismatched, thiol reagents annealed anywhere on the templates examined thus far (up to bases away from the reactive group on the template). Reactants annealed one base away react with similar rates as those annealed 2, 3, 4, 6, 8, 10, 15, 20, or 30 bases away (Fig. In all cases, templated reaction rates are several hundred-fold higher than the rate of untemplated (mismatched) reaction (kapp 104-105 M-ls vs. 5 x 101 At intervening distances of bases, products are efficiently formed presumably through transition states resembling 200membered rings. These findings contrast sharply with the well-known difficulty of 31 of 88 WO 02/074929 PCT/US02/08546 00 O macrocyclization (see, for example, G. Illuminati et al. Acc. Chem. Res. 1981, 14, 95-102; R. B.
Woodward et al. J Am. Chem. Soc. 1981, 103, 3210-3213) in organic synthesis.
[00126] To determine the basis of the distance independence of DNA-templated synthesis, a CC series of modified E templates were first synthesized in which.the intervening bases were replaced by a series of DNA analogs designed to evaluate the possible contribution of (i) interbase interactions, (ii) conformational preferences of the DNA backbone, (iii) the charged (C phosphate backbone, and (iv) backbone hydrophilicity. Templates in which the intervening 0 bases were replaced with any of the analogs in Fig. 9 had little effect on the rates of product 00 Sresponsible for the observed distance independence of DNA-templated synthesis. However, the addition of a 10-base DNA oligonucleotide "clamp" complementary to the single-stranded intervening region significantly reduced product formation (Fig. suggesting that the flexibility of this region is critical to efficient DNA-templated synthesis.
[00127] The distance independent reaction rates may be explained if the bond-forming events in a DNA-templated format are sufficiently accelerated relative to their nontemplated counterparts such that DNA annealing, rather than bond formation, is rate-determining. If DNA annealing is at least partially rate limiting, then the rate of product formation should decrease as the concentration of reagents is lowered because annealing, unlike templated bond formation, is a bimolecular process. Decreasing the concentration of reactants in the case of the E template with one or ten intervening bases between reactive groups resulted in a marked decrease in the observed reaction rate (Fig. 10). This observation suggests that proximity effects in DNAtemplated synthesis can enhance bond formation rates to the point that DNA annealing becomes rate-determining.
[00128] These findings raise the possibility of using DNA-templated synthesis to translate in one pot libraries of DNA into solution-phase libraries of synthetic molecules suitable for PCR amplification and selection. The ability of DNA-templated synthesis to support a variety of transition state geometries suggests its potential in directing a range of powerful watercompatible synthetic reactions (see, Li, C.J. Organic Reactions in Aqueous Media, Wiley and Sons, New York: 1997). The sequence specificity described above suggests that mixtures of reagents may be able to react predictably with complementary mixtures of templates. Finally, the observed distance independence suggests that different regions of DNA "codons" may be 32 of 88 00 used to encode different groups on the same synthetic scaffold without impairing reactions rates.
As a demonstration of this approach, a library of 1,025 maleimide-linked templates was Cc syntheisized, each with a different DNA sequence in an eight-base encoding region (Fig. 11A).
One of these sequences, 5'-TGACGGGT-3', was arbitrarily chosen to code for the attachment of a biotin group to the template. A library of thiol reagents linked to 1,025 different oligonucleotides was also generated. The reagent linked to 3'-ACTGCCCA-5' contained a biotin group, while the other 1,024 reagents contained no biotin. Equimolar ratios of all 1,025 00 templates and 1,025 reagents were mixed in one pot for 10 minutes at 25 "C and the resulting products were selected in vitro for binding to streptavidin. Molecules surviving the selection were amplified by PCR and analyzed by restriction digestion and DNA sequencing.
[00129] Digestion with the restriction endonuclease Tsp45I, which cleaves GTGAC and therefore cuts the biotin encoding template but none of the other templates, revealed a 1:1 ratio of biotin encoding to non-biotin encoding templates following selection (Fig. 12). This represents a 1,000-fold enrichment compared with the unselected library. DNA sequencing of the PCR amplified pool before and after selection suggested a similar degree of enrichment and indicated that the biotin-encoding template is the major product after selection and amplification (Fig. 12). The ability of DNA-templated synthesis to support the simultaneous sequence-specific reaction of 1,025 reagents, each of which faces a 1,024:1 ratio of non-partner to partner templates, demonstrates its potential as a method to create synthetic libraries in one pot. The above proof-of-principle translation, selection, and amplification of a synthetic library member having a specific property (avidin affinity in this example) addresses several key requirements for the evolution of non-natural small molecule libraries toward desired properties.
[00130] Taken together, these results suggest that DNA-templated synthesis is a surprisingly general phenomenon capable of directing, rather than simply encoding, a range of chemical reactions to form products unrelated in structure to nucleic acid backbones. For several reactions examined, the DNA-templated format accelerates the rate of bond formation beyond the rate of a DNA oligonucleotide annealing to its complement, resulting in surprising distance independence. The facile nature of long-distance DNA-templated reactions may also arise in part from the tendency of water to contract the volume of nonpolar reactants (see, Li et al.
Organic Reactions in Aqueous Media, Wiley and Sons: New York, 1997) and from possible compactness of the intervening single-stranded DNA between reactive groups. These findings 33 of 88 WO 02/074929 PCT/US02/08546 00 o may have implications for prebiotic evolution and for understanding the mechanisms of catalytic nucleic acids, which typically localize substrates to a strand of RNA or DNA.
o3 [00131] Methods: S[00132] DNA synthesis. DNA oligonucleotides were synthesized on a PerSeptive Biosystems Expedite 8909 DNA synthesizer using standard protocols and purified by reverse phase HPLC. Oligonucleotides were quantitated spectrophotometrically and by denaturing NC polyacrylamide gel electrophoresis (PAGE) followed by staining with ethidium bromide or SYBR Green (Molecular Probes) and quantitation using a Stratagene Eagle Eye II densitometer.
SPhosphoramidites enabling the synthesis of 5'-NH 2 -dT, 5' tetrachlorofluorescein, abasic 0 0 backbone spacer, C3 backbone spacer, 9-bond polyethylene glycol spacer, 12-bond saturated Shydrocarbon spacer, and 5' biotin groups were purchased from Glen Research. Thiol-linked oligonucleotide reagents were synthesized on C3 disulfide controlled pore glass (Glen Research).
[00133] Template functionalization. Templates bearing 5'-NH 2 -dT groups were transformed into a variety of electrophilic functional groups by reaction with the appropriate electrophile- NHS ester (Pierce). Reactions were performed in 200 mM sodium phosphate pH 7.2 with 2 mg/mL electrophile-NHS ester, 10% DMSO, and up to 100 [g of 5'-amino template at 25 °C for 1 h. Desired products were purified by reverse-phase HPLC and characterized by gel electrophoresis and MALDI mass spectrometry.
[00134] DNA-templated synthesis reactions. Reactions were initiated by mixing equimolar quantities of reagent and template in buffer containing 50 mM MOPS pH 7.5 and 250 mM NaC1 at the desired temperature (25 °C unless stated otherwise). Concentrations of reagents and templates were 60 nM unless otherwise indicated. At various time points, aliquots were removed, quenched with excess P-mercaptoethanol, and analyzed by denaturing PAGE.
Reaction products were quantitated by densitometry using their intrinsic fluorescence or by staining followed by densitometry. Representative products were also verified by MALDI mass spectrometry.
[00135] In vitro selection for avidin binding. Products of the library translation reaction were isolated by ethanol precipitation and dissolved in binding buffer (10 mM Tris pH 8, 1 M NaCI, 10 mM EDTA). Products were incubated with 30 ig of streptavidin-linked magnetic beads (Roche Biosciences) for 10 min at room temperature in 100 uL total volume. Beads were washed 16 times with binding buffer and eluted by treatment with 1 jmol free biotin in 100 uL 34 of 88 WO 02/074929 PCT/US02/08546 00
O
O binding buffer at 70 OC for 10 minutes. Eluted molecules were isolated by ethanol precipitation and amplified by standard PCR protocols (2 mM MgC12, 55 °C annealing, 20 cycles) using the primers 5'-TGGTGCGGAGCCGCCG and C TCGGCTCGG. Automated DNA sequencing used the primer
CCACTGTCCGTGGCGCGACCC.
[00136] DNA Sequences. Sequences not provided in the figures are as follows: matched C, reagent in Fig. 6b SIAB and SBAP reactions: Smismatched reagent in Fig. 6b SIAB and SBAP reactions: C,1 SH; mismatched reagents for other reactions in Figs. 6b, 6c, 6d, and 8a; 00 SSH tetrachlorofluorescein); reagents in Figs. 6c and 6d containing one mismatch: SFAATTCTTACC-SH; E templates in Figs. 6a, 6b SMCC, GMBS, BMPS, and SVSB reactions, and 8a: '5'-(NH 2 dT)-
CGCGAGCGTACGCTCGCGATGGTACGAATTCGACTCGGGAATACCACCTTCGACTCG
AGG; H template in Fig. 6b SIAB, SBAP, and SIA reactions: 5'-(NH 2 dT)- CGCGAGCGTACG CTCGCG ATGGTACGAATTC; clamp oligonucleotide in Fig 8b: [00137] Example 2: Exemplary Reactions for Use in DNA-Templated Synthesis: [00138] As discussed above, the generality of DNA-templated synthetic chemistry was examined (see, Liu et al. J Am. Chem. Soc. 2001, 123, 6961). Specifically, the ability of DNAtemplated synthesis to direct a modest collection of chemical reactions without requiring the precise alignment of reactive groups into DNA-like conformations was demonstrated. Indeed, the distance independence and sequence fidelity of DNA-templated synthesis allowed the simultaneous, one-pot translation of a model library of more than 1,000 templates into the corresponding thioether products, one of which was enriched by in vitro selection for binding to the protein streptavidin and amplified by PCR.
[00139] As described in detail herein, the generality of DNA-templated synthesis has been further expanded and it has been demonstrated that a variety of chemical reactions can be utilized for the construction of small molecules and in particular, for the first time, DNAtemplated organometallic couplings and carbon-carbon bond forming reactions other than pyrimidine photodimerization. These reactions clearly represent an important step towards the in of 88 00
O
vitro evolution of non-natural synthetic molecules by enabling the DNA-templated construction of a much more diverse set of structures than has previously been achieved.
M [00140] The ability of DNA-templated synthesis to direct reactions that require a non-DNAlinked activator, catalyst or other reagent in addition to the principal reactants has also been demonstrated herein. To test the ability of DNA-templated synthesis to mediate such reactions without requiring structural mimicry of the DNA-templated backbone, DNA-templated reductive aminations between an amine-linked template and benzaldehyde- or glyoxal-linked reagents 00 with millimolar concentrations of NaBH 3 CN at room temperature in aqueous solutions can be performed. Significantly, products formed efficiently when the template and reagent sequences were complementary, while control reactions in which the sequence of the reagent did not complement that of the template, or in which NaBH 3 CN was omitted, yielded no significant product (see Figures 13A-13D and 14). Although DNA-templated reductive aminations to generate products closely mimicking the structure of double-stranded DNA have been previously reported (see, for example, X. Li et al. J. Am. Chem. Soc. 2002, 124, 746 and Y. Gat et al.
Biopolymers 1998, 48, 19), the above results demonstrate that reductive amination to generate structures unrelated to the phosphoribose backbone can take place efficiently and sequencespecifically. Referring to Figures 15A and 15B, DNA-templated aide bond formations between amine-linked templates 4 and 5 and carboxylate-linked reagents 6-9 mediated by 1-(3dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxylsulfosuccinimide (sulfo- NHS) to generate amide products in good yields at pH 6.0, 25 0 C (Figures 15A and Product formation was sequence-specific, dependent on the presence of EDC, and suprisingly insensitive to the steric encumbrance of the amine or carboxylate. Efficient DNA-templated amide formation was also mediated by the water-stable activator 4-(4,6-dimethoxy-l,3,5-trizin- 2-yl)-4-methylmorpholinium chloride (DMT-MM) instead of EDC and sulfo-NHS (Figures 14 and 15B). The efficiency and generality of DNA-templated amide bond formation under these conditions, together with the large number of commercially available chiral amines and carboxylic acids, make this reaction an attractive candidate in future DNA-templated syntheses of structurally diverse small molecule libraries.
[00141] It will be appreciated that carbon-carbon bond forming reactions are also important in both chemical and biological syntheses and thus several such reactions are utilized in DNAtemplated format. Both the reaction of nitroalkane-linked reagent (10) with aldehyde-linked template (11) (nitro-aldol or Henry reaction) and the conjugate addition of 10 to maleimideis 36 of 88 00
O
linked template (12) (nitro-Michael addition) proceeded efficiently and with high sequence c specificity at pH 7.5-8.5, 25°C (Figures 13A and 14). In addition, the sequence-specific DNA- S templated Wittig reaction between stabilized phosphorus ylide reagent 13 and aldehyde-linked templates 14 or 11 provided the corresponding olefin products in excellent yields at pH 6.0-8.0, 0 C (Figures 13B-13C and 14). Similarly, the DNA templated 1,3-dipolar cycloaddition C between nitrone-linked reagents 15 and 16 and olefin-linked templates 12, 17 or 18 also afforded products sequence specifically at pH 7.5, 25 0 C (Figures 13B-13C and 14).
1 [00142] In addition to the reactions described above, organometallic coupling reactions can 0 also be utilized in the present invention. For example, DNA-templated Heck reactions were performed in the presence of water-soluble Pd precatalysts. In the presence of 170 mM Na 2 PdC14, aryl iodide-linked reagent 19 and a variety of olefin-linked templates including maleimide 12, acrylamide 17, vinyl sulfone 18 or cinnamamide 20 yielded Heck coupling products in modest yields at pH 5.0, 25 0 C (Figures 13D and 14). For couplings with olefins 17, 18 and 20, adding two equivalents of P(p-S0 3
C
6
H
4 3 per equivalent of Pd prior to template and reagent addition typically increased overall yields by 2-fold. Control reactions containing sequence mismatches or lacking Pd precatalyst yielded no product. To our knowledge, the above DNA-templated nitro aldol addition, nitro Michael addition, Wittig olefination, dipolar cycloaddition, and Heck coupling represent the first reported nucleic-acid templated organometallic reactions and carbon-carbon bond forming reactions other than pyrimidine photodimerization.
[00143] It was previously discovered that the same DNA-templated reactions demonstrate distance independence, the ability to form product at a rate independent of the number of intervening bases between annealed reactants. It was hypothesized (Figure 16a) that distance independence arises when the rate of bond formation in the DNA-templated reaction is greater than the rate of template-reagent annealing. Although only a subset of chemistries fall into this category, any DNA-templated reaction that affords comparable product yields when the reagent is annealed at various distances from the reactive end of the template is of special interest because it can be encoded at a variety of template positions. To evaluate the ability of the DNAtemplated reactions developed above to take place efficiently when reactants are separated by distances relevant to library encoding, the yields of reductive amination, amide formation, nitroaldol addition, nitro-Michael addition, Wittig olefination, dipolar cycloaddition, and Heck 37 of 88 WO 02/074929 PCT/US02/08546 00 O coupling when zero or ten bases separated annealed reactive groups (Figure 16a, n=0 versus were compared. Among the reactions described above or in previous work, amide bond Sformation, nitro-aldol addition, Wittig olefination, Heck coupling, conjugate addition of thiols to CM maleimides and SN 2 reaction between thiols and a-iodo amides demonstrate comparable product formation when reactive groups are separated by zero or ten bases (Figure 16b). These findings indicate that these reactions can be encoded during synthesis by nucleotides that are distal from C1 the reactive end of the template without significantly impairing product formation.
S[00144] In addition to the DNA-templated SN 2 reaction, conjugate addition, vinyl sulfone C addition, amide bond formation, reductive amination, nitro-aldol (Henry reaction), nitro Michael, 00 Wittig olefination, 1,3-dipolar cycloaddition and Heck coupling reactions described directly above, a variety of additional reagents can also be utilized in the method of the present invention.
For example, as depicted in Figure 17, powerful aqueous DNA-templated synthetic reactions including, but not limited to, the Lewis acid-catalyzed aldol addition, Mannich reaction, Robinson annulation reactions, additions of allyl indium, zinc and tin to ketones and aldehydes, Pd-assisted allylic substitution, Diels-Alder cycloadditions, and hetero-Diels-Alder reactions can be utilized efficiently in aqueous solvent and are important complexity-building reactions.
[00145] Taken together, these results expand considerably the reaction scope of DNAtemplated synthesis. A wide variety of reactions proceeded efficiently and selectively only when the corresponding reactants are programmed with complementary sequences. By augmenting the repertoire of known DNA-templated reactions to now include carbon-carbon bond forming and organometallic reactions (nitro-aldol additions, nitro-Michael additions, Wittig olefinations, dipolar cycloadditions, and Heck couplings) in addition to previously reported amide bond formation (see, Schmidt et al. Nucleic Acids Res. 1997, 25, 4792; Bruick et al. Chem. Biol. 1996, 3, 49), imine formation (Czlapinski et al. J. Am. Chem. Soc. 2001, 123, 8618), reductive amination (Li et al. J. Am. Chem. Soc. 2002, 124, 746; Gat et al. Biopolymers, 1998, 48, 19), SN2 reactions (Gartner et al. J. Am. Chem. Soc. 2001, 123, 6961; Xu et al. Nat. Biotechnol. 2001, 19, 148; Herrlein et al. J. Am. Chem. Soc. 1995, 117, 10151) conjugate addition ofthiols (Gartner et al. J. Am. Chem. Soc. 2001, 123, 6961), and phosphoester or phosphonamide formation (Orgel et al. Acc. Chem. Res. 1995, 28, 109; Luther et al. Nature, 1998, 396, 245), these results may enable the sequence-specific translation of libraries of DNA into libraries of structurally and functionally diverse synthetic products. Since minute quantities of templates encoding desired 38 of 88 00 molecules can be amplified by PCR, the yields of DNA-templated reactions are arguably less critical than the yields of traditional synthetic transformations. Nevertheless, many of the Cc reactions developed above proceed efficiently. In addition, by demonstrating that DNAtemplated synthesis in the absence of proteins can direct a large diversity of chemical reactions, these findings support previously proposed hypotheses that nucleic acid-templated synthesis may have translated replicable information into some of the earliest functional molecules such as polyketides, terpenes and polypeptides prior to the evolution of protein-based enzymes. The 00 diversity of chemistry shown here to be controllable simply by bringing reactants into proximity by DNA hybridization without obvious structural requirements provides an experimental basis for these possibilities. The translation of amplifiable information into a wide range of structures is a key requirement for applying nature's molecular evolution approach to the discovery of nonnatural molecules with new functions.
[00146] Methods for Exemplary Reactions for Use in DNA-Templated Synthesis: [00147] Functionalized templates and reagents were typically prepared by reacting 5'-NH 2 terminated oligonucleotides (for template 5'-NH 2
-(CH
2 0) 2 terminated oligonucleotides (for all other templates) or 3'-OP0 3
-CH
2
CH(CH
2 0H)(CH 2 4
NH
2 terminated nucleotides (for all reagents) with the appropriate NHS esters (0.1 volumes of a 20 mg/mL solution in DMF) in 0.2 M sodium phosphate buffer, pH 7.2, 25 0 C, 1 h to provide the template and reagent structures shown in Figures 13A-13D, 15A, and 15B. For amino acid linked reagents 6-9, 3'-
OPO
3
CH
2
CH(CH
2 0H)(CH 2 4
NH
2 terminated oligonucleotides in 0.2 M sodium phosphate buffer, pH 7.2 were reacted with 0.1 volumes of a 100 mM bis[2- (succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES, Pierce) solution in DMF for 10 min at 0 C, followed by 0.3 volumes of a 300 mM amino acid in 300 mM NaOH for 30 min at 25 0
C.
[00148] Functionalized templates and reagents were purified by gel filtration using Sephadex followed by reverse-phase HPLC (0.1 triethylammonium acetate-acetonitrile gradient) and characterized by MALDI mass spectrometry. DNA templated reactions were conducted under the conditions described in Figures 13A-13D, 15A, and 15B and products were characterized by denaturing polyacrylamide gel electrophoresis and MALDI mass spectrometry.
[00149] The sequences of oligonucleotide templates and reagents are as follows to 3' direction, n refers to the number of bases between reactive groups when template and reagent are annealed as shown in Figure 16). 1: TGGTACGAATTCGACTCGGG; 2 and 3 matched: 39 of 88 00 GAGTCGAATTCGTACC; 2 and 3 mismatched: GGGCTCAGCTTCCCCA; 4 and GGTACGAATTCGACTCGGGAATACCACCTT; 6-9 matched (n 10): TCCCGAGTCG; 6 S matched (n AATTCGTACC; 6-9 mismatched: TCACCTAGCA; 11, 12, 14, 17, 18, GGTACGAATTCGACTCGGGA; 10, 13, 16, 19 matched: TCCCGAGTCGAATTCGTACC; S 10, 13 16, 19 mismatched: GGGCTCAGCTTCCCCATAAT; 15 matched: AATTCGTACC; mismatched: TCGTATTCCA; template for n 10 vs. n 0 comparison:
TAGCGATTACGGTACGAATTCGACTCGGGA.
S [100150] Reaction yields quantitated by denaturing polyacrylamide gel electrophoresis followed by ehidium bromide staining, UV visualization, and CCD-based densitometry of product and template starting material bands. Yield calculations assumed that templates and products stained with equal intensity per base; for those cases in which products are partially double-stranded during quantitation, changes in staining intensity may result in higher apparent yields.
[00151] Example 3: Development of Exemplary Linkers [00152] As will be appreciated by one of ordinary skill in the art, it is frequently useful to leave the DNA moiety of the reagents linked to products during reaction development to facilitate analysis by gel electrophoresis. The use of DNA-templated synthesis to translate libraries of DNA into corresponding libraries of synthetic small molecules suitable for in vitro selection, however, requires the development of cleavable linkers connecting reactive groups of reagents with their decoding DNA oligonucleotides. As described below and herein, three exemplary types of linkers have been developed (see, Figure 18). For reagents with one reactive group, it would be desirable to position DNA as a leaving group to the reactive moiety. Under this "autocleavable" linker strategy, the DNA-reactive group bond is cleaved as a natural consequence of the reaction. As but one example of this approach, a fluorescent Wittig phosphorane reagent (14, referring to Figure 19) was synthesized in which the decoding DNA oligonucleotide was attached to one of the aryl phosphine groups (see, Figure 19, left). DNAtemplated Wittig reaction with aldehyde-linked templates resulted in the nearly quantitative transfer of the fluorescent group from the Wittig reagent to the template and the concomitant liberation of the alkene product from the DNA moiety of the reagent. Additionally, reagents bearing more than one reactive group can be linked to their decoding DNA oligonucleotides through one of two additional linker strategies. In the "scarless" linker strategy, DNA-templated of 88 WO 02/074929 PCT/US02/08546 00 0 O reaction of one reactive group is followed by cleavage of the linker attached through a second Sreactive group to yield products without leaving behind additional chemical functionality. For example, a series of amino acid reagents were syntheisized which were connected through a M carbamoylethylsulfone linker to their decoding DNA oligonucleotides (Figure 19, center).
Products of DNA-templated amide bond formation using these amino acid reagents were treated with aqueous alkaline buffer to effect the quantitative elimination and spontaneous decarboxylation of the carbamoyl group. The product of leaving this scarless linker is therefore Sthe cleanly transferred amino acid moiety. In yet other embodiment of the invention, a third C1 linker strategy, a "useful scar" may be utilized on the theory that it may be advantageous to 00 Sintroduce useful chemical groups as a consequence of linker cleavage. In particular, a "useful scar" can be functionalized in subsequent steps and is left behind following linker cleavage. For example, amino acid reagents linked through 1,2-diols to their decoding DNA oligonucleotides were generated. Following amide bond formation, this linker was quantitatively cleaved by oxidation with NaI04 to afford products bearing useful aldehyde groups (see, Figure 19, right).
In addition to the linkers described directly above, a variety of additional linkers can be utilized.
For example, as shown in Figure 20, a thioester linker can be generated by carbodiimidemediated coupling of thiol-terminated DNA with carboxylate-containing reagents and can be cleaved with aqueous base. As the carboxylate group provides entry into the DNA-templated amide bond formation reactions described above, this linker would liberate a "useful scar" when cleaved (see, Figure 20). Alternatively, the thioester linker can be used as an autocleavable linker during an amine acylation reaction in the presence of Ag(I) cations (see, Zhang et al. J.
Am. Chem. Soc. 1999, 121, 3311-3320) since the thiol-DNA moiety of the reagent is liberated as a natural consequence of the reaction. It will be appreciated that a thioether linker that can be oxidized and eliminated at pH 11 to liberate a vinyl sulfone can be utilized as a "useful scar" linker. As demonstrated herein, the vinyl sulfone group serves as the substrate in a number of subsequent DNA-templated reactions.
[00153] Example 4: Exemplary Reactions in Organic Solvents: [00154] As demonstrated herein, a variety of DNA-templated reactions can occur in aqueous media. It has also been demonstrated, as discussed below, that DNA-templated reactions can occur in organic solvents, thus greatly expanding the scope of DNA-templated synthesis.
Specifically, DNA templates and reagents have been complexed with long chain 41 of 88 WO 02/074929 PCT/US02/08546 00 0 O tetraalkylammonium cations (see, Jost et al. Nucleic Acids Res. 1989, 17, 2143; Mel'nikov et al.
SLangmuir, 1999, 15, 1923-1928) to enable quantitative dissolution of reaction components in h anhydrous organic solvents including CH 2 C1 2 CHC1 3 DMF and MeOH. Surprisingly, it was C found that DNA-templated synthesis can indeed occur in anhydrous organic solvents with high sequence selectivity. Depicted in Figure 21 are DNA-templated amide bond formation reacations in which reagents and templates are complexed with dimethyldidodecylammonium C cations either in separate vessels or after preannealing in water, lyophilized to dryness, dissolved 0in CH 2 C12, and mixed together. Matched, but not mismatched, reactions provided products both C'l when reactants were preannealed in aqueous solution and when they were mixed for the first 00 Stime in CH 2 Cl 2 (see, Figure 21). DNA-templated amide formation and Pd-mediated Heck rK coupling in anhydrous DMF also proceeded sequence-specifically. Clearly, these observations of sequence-specific DNA-templated synthesis in organic solvents implies the presence of at least some secondary structure within tetraalkylammonium-complexed DNA in organic media, and should enable DNA receptors and catalysts to be evolved towards stereoselective binding or catalytic properties in organic solvents. Specifically, DNA-templated reactions that are known to occur in aqueous media, including conjugate additions, cycloadditions, displacement reactions, and Pd-mediated couplings can also be performed in organic solvents. In certain other embodiments, reactions in organic solvents may be utilized that are inefficient or impossible to perform in water. For example, while Ru-catalyzed olefin metathesis in water has been reported by Grubbs and co-workers (see, Lynn et al. J Am. Chem. Soc. 1998, 120, 1627-1628; Lynn et al.
J. Am. Chem. Soc. 2000, 122, 6601-6609; Mohr et al. Organometallics 1996, 15, 4317-4325), the aqueous metathesis system is extremely functional group sensitive. The functional group tolerance of Ru-catalyzed olefin metathesis in organic solvents, however, is significantly more robust. Some exemplary reactions to utilize in organic solvents include, but are not limited to 1,3-dipolar cycloaddition between nitrones and olefins which can proceed through transition states that are less polar than ground state starting materials.
[00155] As detailed above, the generality of DNA-templated synthesis has been established by performing several distinct DNA-templated reaction types, none of which are limited to producing structures that resemble the natural nucleic acid backbone, and many of which are highly useful carbon-carbon bond forming or complexity-building synthetic reactions. It has been shown that the distance independence of DNA-templated synthesis allows different regions 42 of 88 WO 02/074929 PCT/US02/08546 00 0 O of a DNA template to each encode different synthetic reactions. DNA-templated synthesis can Smaintain sequence fidelity even in a library format in which more than 1,000 templates and 1,000 reagents react simultaneously in one pot. As described above and below, linker strategies n have been developed, which together with the reactions developed as described above, have enabled the first multi-step DNA-templated synthesis of simple synthetic small molecules.
Additionally, the sequence-specific DNA-templated synthesis in organic solvents has been dehonstrated, further expanding the scope of this approach.
S[00156] Example 5: Synthesis of Exemplary Compounds and Libraries of Compounds: (Cl [00157] A) Synthesis of a Polycarbamate Library: One embodiment of the strategy 00 described above is the creation of an amplifiable polycarbamate library. Of the sixteen possible (C1 dinucleotides used to encode the library, one is assigned a start codon function, and one is assigned to serve as a stop codon. An artificial genetic code is then created assigning each of the up to 14 remaining dinucleotides to a different monomer. For geometric reasons one monomer actually contains a dicarbamate containing two side chains. Within each monomer, the dicarbamate is attached to the corresponding dinucleotide (analogous to a tRNA anticodon) through a silyl enol ether linker which liberates the native DNA and the free carbamate upon treatment with fluoride. The dinucleotide moiety exists as the activated 5'-2-methylimidazole phosphate, that has been demonstrated (Inoue et al. J Mol. Biol. 162:201, 1982; Rembold et al.
J Mol. Evol. 38:205, 1994; Chen et al. J Mol. Biol. 181:271, 1985; Acevedo et al. J. Mol. Biol.
197:187, 1987; Inoue et al. J. Am. Chem. Soc. 103:7666, 1981; each of which is incorporated herein by reference) to serve as an excellent leaving group for template-directed oligomerization of nucleotides yet is relatively stable under neutral or basic aqueous conditions (Schwartz et al.
Science 228:585, 1985; incorporated herein by reference). The dicarbamate moiety exists in a cyclic form linked through a vinyloxycarbonate linker. The vinylcarbonate group has been demonstrated to be stable in neutral or basic aqueous conditions (Olofson et al. Tetrahedron Lett.
18:1563, 1977; Olofson et al. Tetrahedron Lett. 18:1567, 1977; Olofson et al. Tetrahedron Lett.
18:1571, 1977; each of which is incorporated herein by reference) and further has been shown to provide carbamates in very high yields upon the addition of amines (Olofson et al. Tetrahedron Lett. 18:1563, 1977; incorporated herein by reference).
43 of 88 WO 02/074929 PCT/US02/08546 00
O
O Me leaving group forDNA N N-P c' polymerization I O 0 0k 0 B 2 TBSO
SHO
R 0 00 eN accepts nucleophile for carbamate 0 polymerization; releases a new O \NH nucleophile when 0 attacked [00158] When attacked by an amine from a nascent polycarbamate chain, the vinyl carbonate linker, driven by the aromatization of m-cresol, liberates a free amine. This free amine subsequently serves as the nucleophile to attack the next vinyloxycarbonate, propagating the polymerization of the growing carbamate chain. Such a strategy minimizes the potential for cross-reactivity and bi-directional polymerization by ensuring that only one nucleophile is present at any time during polymerization.
[00159] Using the monomer described above, artificial translation of DNA into a polycarbamate can be viewed as a three-stage process. In the first stage, single stranded DNA templates encoding the library are used to guide the assembly and polymerization of the dinucleotide moieties of the monomers, terminating with the "stop" monomer which possesses a 3'methyl ether instead of a 3'hydroxyl group (Figure 22).
[00160] Once the nucleotides have assembled and polymerized into double-stranded DNA, the "start" monomer ending in a o-nitrobenzylcarbamates is photodeprotected to reveal the primary amine that initiates carbamate polymerization. Polymerization proceeds in the 5' to 3' direction along the DNA backbone, with each nucleophilic attack resulting in the subsequent 44 of 88 WO 02/074929 PCT/US02/08546 00 0 O unmasking of a new amine nucleophile.. Attack of the "stop" monomer liberates an acetamide Srather than an amine, thereby, termination polymerization (Figure 23). Because the DNA at this stage exists in a stable double-stranded form, variables such as temperature and pH may be M explored to optimize polymerization efficiency.
[00161] Following polymerization the polycarbamate is cleaved from the phosphate backbone of the DNA upon treatment with fluoride. Desilylation of the enol ether linker and the C elimination of the phosphate driven by the resulting release of phenol provides the Spolycarbamate covalently linked at its carboxy terminus to its encoding single-stranded DNA C1 (Figure 24).
00 0[00162] At this stage the polycarbamate may be completely liberated from the DNA by base Shydrolysis of the ester linkage. The liberated polycarbamate can be purified by HPLC and retested to verify that its desired properties are intact. The free DNA can be amplified using PCR, mutated with error-prone PCR (Cadwell et al. PCR Methods Appl. 2:28, 1992; incorporated herein by reference) or DNA shuffling (Stemmer Proc. Natl. Acad. Sci. USA 91:10747, 1994; Stemmer Nature 370:389, 1994; U.S. Patent 5,811,238, issued September 22, 1998; each of which is incorporated herein by reference), and/or sequenced to reveal the primary structure of the polycarbamate.
[00163] Synthesis of monomer units. After the monomers are synthesized, the assembly and polymerization of the monomers on the DNA scaffold should occur spontaneously. Shikimic acid 1, available commercially, biosynthetically (Davis Adv. Enzymol. 16:287, 1955; incorporated herein by reference), or by short syntheses from D-mannose (Fleet et al. J. Chem.
Soc., Perkins Trans. 1 905, 1984; Harvey et al. Tetrahedron Lett. 32:4111, 1991; each of which is incorporated herein by reference), serves as a convenient starting point for the monomer synthesis. The syn hydroxyl groups are protected as the p-methoxybenzylidene, and remaining hydroxyl group as the tert-butyldimethylsilyl ether to afford 2. The carboxylate moiety of the protected shikimic acid is then reduced completely by LAH reduction, tosylation of the resulting alcohol, and further reduction with LAH to provide 3.
of 88 WO 02/074929 PCTIUS02/08546 W0 02 00
CO
2 H
CO
2 H Me S1) p-MeOC 6
H
4 CHO, TsOH 1) LAIH 4 S 2) TBSCI, imidazole 2) TsC, pyridine ,HO '/OH 0 "/OTBS 3) LiAI 4 o /OTBS OH 0 0 p-MeOPh p-MeOPh 1 2 3 C [00164] Commercially available and synthetically accessible N-protected amino acids serve as Sthe starting materials for the dicarbamate moiety of each monomer. Reactive side chains are protected as photolabile ethers, esters, acetals, carbamates, or thioethers. Following chemistry 00 previously developed (Cho et al. Science 261:1303, 1993; incorporated herein by reference), a desired amino acid 4 is converted to the corresponding amino alcohol 5 by mixed anhydride formation with isobutylchloroformate followed by reduction with sodium borohydride. The amino alcohol is then converted to the activated carbonate by treatment with pnitrophenylchloroformate to afford 6, which is then coupled to a second amino alcohol 7 to provide, following hydroxyl group silylation and FMOC deprotection, carbamate 8.
0 wn -oc 0 1) i-BuOCOCI NHFMOC 2) NaBH 4 NHFMOC pyridine .NHFMOC HO 2 HO- p-NO 2 PhO O 4 R 5 R 6 RI 1) 0j 2 0 TESONH O NH2 2) TESCI, imid.
3) piperidine 8 [00165] Coupling of carbamate 8 onto the shikimic acid-derived linker proceeds as follows.
The allylic hydroxyl group of 3 is deprotected with TBAF, treated with triflic anhydride to form the secondary triflate, then displaced with aminocarbamate 8 to afford 9. Presence of the vinylic methyl group in 3 should assist in minimizing the amount of undesired product resulting from SN2' addition (Magid Tetrahedron 36:1901, 1980; incorporated herein by reference). Michael additions of deprotonated carbamates to a,P-unsaturated esters have been well documented (Collado et al. Tetrahedron Lett. 35:8037, 1994; Hirama et al. J. Am. Chem. Soc. 107:1797, 46 of 88 Ol\ n/0An 4'2O PCT/TS02/n0d46 00 1985; Nagasaka et al. Heterocycles 29:155, 1989; Shishido et al. J. Chem. Soc. Perkins Trans. I S993, 1987; Hirama et al. Heterocycles 28:1229, 1989; each of which is incorporated herein by reference). By analogy, the secondary amine is protected as the o-nitrobenzyl carbamate (NBOC), and the resulting compound is deprotonated at the carbamate nitrogen. This deprotonation can typically be performed with either sodium hydride or potassium tertbutyloxide (Collado et al. Tetrahedron Lett. 35:8037, 1994; Hirama et al. J Am. Chem. Soc.
S107:1797, 1985; Nagasaka et al. Heterocycles 29:155, 1989; Shishido et al. J. Chem. Soc.
SPerkins Trans. I 993, 1987; Hirama et al. Heterocycles 28:1229, 1989; each of which is c incorporated herein by reference), although other bases may be utilized to minimize 00 0 deprotonation of the nitrobenzylic protons. Additions of the deprotonated carbamate to a,3c1 unsaturated ketone 10, followed by trapping of the resulting enolate with TBSC1, should afford silyl enol ether 11. The previously found stereoselectivity of conjugate additions to enones such as 10 (House et al. J. Org. Chem. 33:949, 1968; Still et al. Tetrahedron 37:3981, 1981; each of which is incorporated herein by reference) suggests that preferential formation of 11 over its diastereomer. Ketone 10, the precursor to the fluoride-cleavable carbamatephosphate linker, may be synthesized from 2 by one pot decarboxylation (Barton et al.
Tetrahedron 41:3901, 1985; incorporated herein by reference) followed by treatment with TBAF, Swern oxidation of the resulting alcohol to afford 12, deprotection with DDQ, selective nitrobenzyl ether formation of the less-hindered alcohol, and reduction of the a-hydroxyl group with samarium iodide (Molander In Organic Reactions, Paquette, Ed. 46:211, 1994; incorporated herein by reference).
47 of 88 WO 02/074929 PCT/US02/08546 00 c-I 1) TBAF 2) Tr 2 O, pydhne
R
3)8 0 OTS0 VM
H
P-M.Oph/ 3 psp I)DMAI', DDQ, 0 '0TS3) TBAF 3) 0 O.12 00CCCC p-OMoOPh> P-W.P, 2 12 BNO#*OB 00 0 N 0 N -O 0 2 C TBCI~ f flooa O9 0 ,11SIP-O~
'IN
[00 1661 The p-methoxybenzylidiene group of 11 is transformed into the a-hydroxy PMB ether using sodium cyanoborohydride and TMS chloride (Johansson et al J Chei. Soc. Perkin Trans.
1 2371, 1984; incorporated herein by reference) and the TES group deprotected with 2% HF (conditions that should not affect the TBS ether (Bosehelli et al Tetrahedron Lett. 26:5239, 1985; incorporated herein by reference)) to provide 13. The PMvB group, following precedent (Johansson et al Chemn. Soc. Perkin Trans. I12371, 1984; Sutherlin et aL Tetrahedron Lett.
34:4897, 1993; each of which is incorporated herein by reference), should remain on the more hindered secondary alcohol. The two free hydroxyl groups may be macrocyclized by very slow addition of 13 to a solution of p-nitrophenyl chioroformate (or another phosgene analog), providing 14. The PMB ether is deprotected, and the resulting alcohol is converted into a triflate and eliminated under kinetic conditions with a sterically hindered base to afford vinyloxycarbonate 15. Photodeprotection of the nitrobeuzyl either and nitrobenzyl carbamate yields alcohol 16.
48 of 88 WO 02/074929 PCT/US02/08546 00
C
6
H
4 OMe rl0 OTES OH OH T 0HCN. ,,,OPMB ,\R O N 2)2%19F S"/N Oy N4,( ,\ONB OY N B 0 "0NB I O0"O1NB 13 OTBS OTBS bn.
0 0 ,R OR
\R
2
ONB
1)TO ODQ 2R W2 N 3)D 00 .hiiBOPM O P TBS 0 0 OTBS lR-NB,R-COZ4BN R'16: R.R4 ON B [00167] The monomer synthesis is completed by the sequential coupling of three components.
Chlorodiisopropylamninophosphine 17 is synthesized by the reaction Of PCi 3 With diisopropylamnine (King et al. Org. Chem. 49:1784, 1984; incorporated herein by reference).
Resin-bound (or 3'-o-nitrobenzylether protected) nucleoside 18 is coupled to 17 to afford phosphoramidite 19. Subsequent coupling of 19 with the nucleoside 20 (Inoue et a. J Am.
Chem. Soc. 103:7666, 1981; incorporated herein by reference) provides 21. Alcohol 16 is then reacted with 21 to yield, after careful oxidation using MCPBA or 12 followed by cleavage from the resin (or photodeprotection), the completed monomer 22. This strategy of sequential coupling of 17 with alcohols has been successfully used to generate phosphates bearing three different alkoxy substituents in excellent yields (Bannwarth et al. Helv. Chim. Acta 70:175, 1987; incorporated herein by reference).
49 of 88 WO 02/074929 PCT/US02/08546 I N(iPr) 2 iIr -Pr 0 0 iPr r N(Pr) Me o. f/ Me N BN' 0 0 20 HO STBSOM 0 e H 2) dHMO N( 7a-0 R 2 00 N(iPr)h [00168] The unique start and stop monomers used to initiate and terminate carbamate polymerization may be synthesized by simple modification of the above scheme.
[00169] B) Evolvable Functionalized Peptide-Nucleic Acids (PNAs): In another embodiment an amplifiable peptide-nucleic acid library is created. Orgel and co-workers have demonstrated that peptide-nucleic acid (PNAs) oligomers are capable of efficient polymerization on complementary DNA or RNA templates (B6hler et al. Nature 376:578, 1995; Schmidt et al.
Nucl. Acids Res. 25:4792, 1997; each of which is incorporated herein by reference). This finding, together with the recent synthesis and characterization of chiral peptide nucleic acids bearing amino acid side chains (-Iaaima et al. Angew. Chem. Int. Ed. Engl. 35:1939-1942, 1996; Ptischl et al. Tetrahedron Lett. 39:4707, 1998; each of which is incorporated herein by reference), allows the union of the polymer backbone and the growing nucleic acid strand into a single structure. In this example, each template consists of a DNA hairpin terminating in a amino group; the solid-phase and solution syntheses of such molecules have been previously described (Uhlmann et al. Angew. Chem. Int. Ed. Engl. 35:2632, 1996; incorporated herein by reference). Each extension monomer consists of a PNA trimer (or longer) bearing side chains containing functionality of interest. An artificial genetic code is written to assign each trinucleotide to a different set of side chains. Assembly, activation (with a carbodiimide and of 88 WO 02/074929 PCT/US02/08546 00
O
O appropriate leaving group, for example), and polymerization of the PNA dimers along the Scomplementary DNA template in the carboxy- to amino-terminal direction affords the unnatural polymer (Figure 20). Choosing a "stop" monomer with a biotinylated N-terminus provides a Cc convenient way of terminating the extension and purifying full-length polymers. The resulting polymers, covalently linked to their encoding DNA, are ready for selection, sequencing, or mutation.
C [00170] The experimental approach towards implementing an evolvable functionalized Speptide nucleic acid library comprises improving and adapting known chemistry for the high 00 C"1 efficiency template-directed polymerization of PNAs; (ii) defining a codon format (length and Scomposition) suitable for PNA coupling of a number of diverse monomers on a complementary Sstrand of encoding DNA free from significant infidelity, framshifting, or spurious initiation of polymerization; (iii) choosing an initial set of side chains defining our new genetic code and synthesizing corresponding monomers; and (iv) subjecting a library of functionalized PNAs to cycles of selection, amplification, and mutation and characterizing the resulting evolved molecules to understand the basis of their novel activities.
[00171] Improving coupling chemistry: While Orgel and coworkers have reported template-directed PNA polymerization, reported yields and number of successful couplings are significantly lower than would be desired. A promising route towards improving this key coupling process is exploring new coupling reagents, temperatures, and solvents which were not previously investigated (presumably because previous efforts focused on conditions which could have existed on prebiotic earth). The development of evolvable functionalized PNA polymers involves employing activators (DCC, DIC, EDC, HATU/DIEA, HBTU/DIEA, ByBOP/DIEA, chloroacetonitrile), leaving groups (2-methylimidazole, imidazole, pentafluorophenol, phenol, thiophenol, trifluoroacetate, acetate, toluenesulfonic acids, coenzyme A, DMAP, ribose), solvents (aqueous at several pH values, DMF, DMSO, chloroform, TFE), and temperature (0°C, 4°C, 25°C, 37 0 C, 55 0 C) in a large combinatorial screen to isolate new coupling conditions. Each well of a 384-well plate is assigned a specific combination of one activator, leaving group, solvent, and temperature. Solid-phase synthesis beads covalently linked to DNA hairpin templates are placed in each well, together with a fluorescently labeled PNA monomer complementary to the template. A successful coupling event results in the covalent linking of the fluorophore to the beads (Figure 26); undesired non-templated coupling can be distinguished 51 of 88 WO 02/074929 PCT/US02/08546 00 by control reactions with mismatched monomers. Following bead washing and cleavage of the product from solid support, each well is assayed with a fluorescence plate reader.
[00172] (ii) Defining a codon format: While Nature has successfully employed a triplet C codon in protein biosynthesis, a new polymer assembled under very different conditions without the assistance of enzymes may require an entirely novel codon format. Frameshifting may be remedied by lengthening each codon such that hybridizing a codon out of frame guarantees a Cr mismatch (for example, by starting each codon with a G and by restricting subsequent positions in the codon to T, C, and Thermodynamically, one would also expect fidelity to improve as 00 able to hybridize despite mismatched bases and moreover complicate monomer synthesis. An C1 optimal codon length for high fidelity artificial translation can be defined using an optimized plate-based combinatorial screen developed above. The length and composition of each codon in the template is varied by solid-phase synthesis of the appropriate DNA hairpin. These template hairpins are then allowed to couple with fluorescently labeled PNA monomers of varying sequence. The ideal codon format allows only monomers bearing exactly complementary sequences to couple with templates, even in the presence of mismatched PNA monomers (which are labeled differently to facilitate assaying of matched versus mismatched coupling). Triplet and quadruplet codons in which two bases are varied among A, T, and C while the remaining base or bases are fixed as G to ensure proper registration during polymerization are first studied.
[00173] (iii) Writing a new genetic code: Side chains are chosen which provide interesting functionality not necessarily present in natural biopolymers, which are synthetically accessible, and which are compatible with coupling conditions. For example, a simple genetic code which might be used to evolve a Ni 2 chelating PNA consists of a variety of protected carboxylatebearing side chains as well as a set of small side chains to equip polymers with conformational flexibility and structural diversity (Figure 27). Successful selection of PNAs capable of binding Ni 2 with high affinity could be followed by an expansion of this genetic code to include a fluorophore as well as a fluorescent quencher. The resulting pdlymers could then be evolved towards a fluorescent Ni 2 sensor which possesses different fluorescent properties in the absence or presence of nickel. Replacing the fluorescent side chain with a photocaged one may allow the evolution of polymers that chelate Ni+ 2 in the presence of certain wavelengths of light or which release Ni 2 upon photolysis. These simple examples demonstrate the tremendous flexibility in 52 of 88 WO 02/074929 PCT/US02/08546 00 0 O potential chemical properties of evolvable unnatural molecules conferred by the freedom to Sincorporate synthetic building blocks no longer limited to those compatible with the ribosomal machinery.
C [00174] (iv) Selecting for desired unnatural polymers: Many of the methods developed for the selection of biological molecules can be applied to selections for evolved PNAs with desired properties. Like nucleic acid or phage-display selections, libraries of unnatural polymers C generated by the DNA-templated polymerization methods described above are self-tagged and Stherefore do not need to be spatially separated or synthesized on pins or beads. Ni 2 binding C1 PNA may be done simply by passing the entire library resulting from translation or a random 00 0oligonucleotide through commercially available Ni-NTA ("His-Tag") resin precharged with nickel. Desired molecules bind to the resin and are eluted with EDTA. Sequencing these PNAs after several cycles of selection, mutagenesis, and amplification reveals which of the initially chosen side chains can assemble together to form a Ni+2 receptor. In addition, the isolation of a PNA Ni 2 chelator represents the PNA equivalent of a histidine tag which may prove useful for the purification of subsequent unnatural polymers. Later efforts will involve more ambitious selections. For example, PNAs that fluoresce in the presence of specific ligands may be selected by FACS sorting of translated polymers linked through their DNA templates to beads. Those beads that fluoresce in the presence, but not in the absence, of the target ligand are isolated and characterized. Finally, the use of a biotinylated "stop" monomer as described above allows for the direct selection for the catalysis of many bond-forming or bond-breaking reactions. Two examples depicted in Figure 28 outline a selection for a functionalized PNA that catalyzes the retroaldol cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolysis, as well as a selection for PNA that catalyzes the converse aldol reaction.
[00175] C) Evolvable Libraries of Small Molecules: In yet another embodiment of the present invention, the inventive methods are used in preparing amplifiable and evolvable unnatural nonpolymeric molecules including synthetic drug scaffolds. Nucleophilic or electrophilic groups are individually unmasked on a small molecule scaffold attached by simple covalent linkage or through a common solid support to an encoding oligonucleotide template.
Electrophilic or nucleophilic reactants linked to short nucleic acid sequences are hybridized to 53 of 88 WO 02/074929 PCT/US02/08546 00
O
0 the corresponding templates. Sequence-specific reaction with the appropriate reagent takes place by proximity catalysis (Figure 29).
[00176] Following synthetic functionalization of all positions in a manner determined by the sequence of the attached DNA (Figures 30 31), the resulting encoded beads may be subjected to wide range of biological screens which have been developed for assaying compounds on resin.
(Gordon et al. J Med. Chem. 37:1385, 1994; Gallop et al. J. Med. Chem. 37:1233-1251, 1994; CN each of which is incorporated herein by reference) 0[00177] Encoding DNA is cleaved from each bead identified in the screen and subjected to PCR, mutagenesis, sequencing, or homologous recombination before reattachment to a solid 00 Ssupport. Ultimately, this system is most flexible when the encoding DNA is directly linked to the combinatorial synthetic scaffold without an intervening bead. In this case, entire libraries of compounds may be screened or selected for desired activities, their encoding DNA liberated, amplified, mutated, and recombined, and new compounds synthesized all in a small series of one-pot, massively parallel reactions. Without a bead support, however, reactivities of hybridized reactants must be highly efficient since only one template molecule directs the synthesis of the entire small molecule.
[00178] The development of evolvable synthetic small molecule libraries relies on chemical catalysis provided by the proximity of DNA hybridized reactants. It will be appreciated that acceptable distances between hybridized reactants and unmasked reactive groups must first be defined for efficient DNA-templated functionalization by hybridizing radiolabeled electrophiles (activated esters in out first attempts) attached to short oligonucleotides at varying distances from a reactive nucleophile (a primary amine) on a strand of DNA. At given timepoints, aliquots are subjected to gel electrophoresis and autoradiography to monitor the course of the reaction.
Plotting the reaction as a function of the distance (in bases) between the nucleophile and electrophile will define an acceptable distance window within which proximity-based catalysis of a DNA-hybridized reaction can take place. The width of this window will determine the number of distinct reactions we can encode on a strand of DNA (a larger window allows more reactions) as well as the nature of the codons (a larger window is required for longer codons) (Figure 32).
[00179] Once acceptable distances between functional groups on a combinatorial synthetic scaffold and hybridizes reactants is determined, the codon format is determined. The 54 of 88 WO 02/074929 PCT/US02/08546 00 O nonpolymeric nature of small molecule synthesis simplifies codon reading as frameshifting is not San issue and relatively large codons may be used to ensure that each set of reactants hybridizes only to one region of the encoding DNA strand.
[00180] Once the distance of the linker between the functional group and synthetic small molecule scaffold and the codon format have been determined, one can synthesize small imolecules based on a small molecule scaffold such as the cephalosporin scaffold shown in Figure 31. The primary amine of 7-aminocephalosporanic acid is first protected using FMOC- SCl, and then the acetyl group is hydrolyzed by treatment with base. The encoding DNA template C is then attached through an amide linkage using EDC and HOBt to the carboxylic acid group. A Stransfer molecule with an anti-codon capable of hybridizing to the attached DNA template is r, then allowed to hybridize to the template. The transfer molcule has associated with it through a disulfide linkage a primary amine bearing R 1 Activation of the primary hydroxyl group on the cephalosporin scaffold with DSC following treatment with TCEP affords the amine covalently attached to the scaffold through a carbamate linkage. Further treatment with another transfer unit having an amino acid leads to functionaliztion of the deprotected primary amine of the cephalosporin scaffold. Cephalosporin-like molecules synthesized in this manner may then be selected, amplified, and/or evolved using the inventive methods and compositions. The DNA template may be diversified and evolved using DNA shuffling.
[00181] D) Multi-Step Small Molecule Synthesis Programmed by DNA Templates: Molecular evolution requires the sequence-specific translation of an amplifiable information carrier into the structures of the evolving molecules. This requirement has limited the types of molecules that have been directly evolved to two classes, proteins and nucleic acids, because only these classes of molecules can be translated from nucleic acid sequences. As described generally above, a promising approach to the evolution of molecules other than proteins and nucleic acids uses DNA-templated synthesis as a method of translating DNA sequences into synthetic small molecules. DNA-templated synthesis can direct a wide variety of powerful chemical reactions with high sequence-specificity and without requiring structural mimicry of the DNA backbone. The application of this approach to synthetic molecules of useful complexity, however, requires the development of general methods to enable the product of a DNA-templated reaction to undergo subsequent DNA-templated transformations. The first DNA-templated multi-step small molecule syntheses is described in detail herein. Together with of 88 00
O
recent advances in the reaction scope of DNA-templated synthesis, these findings set the stage for the in vitro evolution of synthetic small molecule libraries.
Cc [00182] Multi-step DNA-templated small molecule synthesis faces two major challenges
O
beyond those associated with DNA-templated synthesis in general. First, the DNA used to direct reagents to appropriate templates must be removed from the product of a DNA-templated reaction prior to subsequent DNA-templated synthetic steps in order to prevent undesired hybridization to the template. Second, multi-step synthesis often requires the purification and 00 isolation of intermediate products, yet common methods used to purify and isolate reaction products are not appropriate for multi-step synthesis on the molecular biology scale. To address these challenges, three distinct strategies were implemented in solid-phase organic synthesis, for linking chemical reagents with their decoding DNA oligonucleotides and two general approaches for product purification after any DNA-templated synthetic step were developed.
[00183] When possible, an ideal reagent-oligonucleotide linker for DNA-templated synthesis positions the oligonucleotide as a leaving group of the reagent. Under this "autocleaving" linker strategy, the oligonucleotide-reagent bond is cleaved as a natural chemical consequence of the reaction (Figs. 33A and 33B). As the first example of this approach applied to DNA-templated chemistry, a dansylated Wittig phosphorane reagent was synthesized in which the decoding DNA oligonucleotide was attached to one of the aryl phosphine groups Hughes, Tetrahedron Lett. 1996, 37, 7595). DNA-templated Wittig olefination (as described above) with aldehydelinked template 2 resulted in the efficient transfer of the fluorescent dansyl group from the reagent to the template to provide olefin 3 (Fig. 33A). As a second example of an autocleaving linker, DNA-linked thioester 4, when activated with Ag(I) at pH 7.0 (Zhang et al. J. Am. Chem.
Soc. 1999, 121, 3311) acylated amino-terminated template 5 to afford amide product 6 (Fig.
33A). Ribosomal protein biosynthesis uses aminoacylated tRNAs in a similar autocleaving linker format to mediate RNA-templated peptide bond formation. To purify desired products away from unreacted reagents and from cleaved oligonucleotides following DNA-templated reactions using autocleaving linkers, biotinylated reagent oligonucleotides and washing crude reactions with streptavidin-linked magnetic beads (Fig. 34) were utilized. Although this approach does not separate reacted templates from unreacted templates, unreacted templates can be removed in subsequent DNA-templated reaction and purification steps (see below).
56 of 88 00
O
[00184] Reagents bearing more than one functional group can be linked to their decoding ct DNA oligonucleotides through a second and third linker strategies. In the "scarless linker" S approach, one functional group of the reagent is reserved for DNA-templated bond formation, while the second functional group is used to attach a linker that can be cleaved without introducing additional unwanted chemical functionality. DNA-templated reaction is followed by cleavage of the linker attached through the second functional group to afford desired products (Fig. 33A). For example, a series of aminoacylation reagents such as (D)-Phe derivative 7 were 00 synthesized in which the a-amine is connected through a carbamoylethylsulfone linker (Zarling et al. J. Immunology 1980, 124, 913) to its decoding DNA oligonucleotide. The product of DNA-templated amide bond formation (as described herein) using this reagent and an amineterminated template was treated with aqueous base to effect the quantitative elimination and spontaneous decarboxylation of the linker, affording product 9 containing the cleanly transferred amino acid group (Fig. 33A). This sulfone linker is stable in pH 7.5 or lower buffer at 25 °C for more than 24 h yet undergoes quantitative cleavage when exposed to pH 11.8 buffer for 2 h at 37
"C.
[00185] In some cases it may be advantageous to introduce new chemical groups as a consequence of linker cleavage. Under a third linker strategy, linker cleavage generates a "useful scar" that can be functionalized in subsequent steps. As an example of this class of linker, amino acid reagents such as the (L)-Phe derivative 10 were generated linked through 1,2diols (Fruchart et al Tetrahedron Lett. 1999, 40, 6225) to their decoding DNA oligonucleotides.
Following DNA-templated amide bond formation with amine terminated template this linker was quantitatively cleaved by oxidation with 50 mM aqueous NaI0 4 at pH 5.0 to afford product 12 containing an aldehyde group appropriate for subsequent functionalization (for example, in a DNA-templated Wittig olefination, reductive amination, or nitrolaldol addition (Fig. 33A).
[00186] Desired products generated from DNA-templated reactions using the scarless or useful scar linkers can be readily purified using biotinylated reagent oligonucleotides (Fig. 34).
Reagent oligonucleotides together with desired products are first captured on streptavidin-linked magnetic beads. Any unreacted template bound to reagent by base pairing is removed by washing the beads with buffer containing 4 M guanidinium chloride. Biotinylated molecules remain bound to the streptavidin beads under these conditions. Desired product is then isolated 57 of 88 17^- ft^ lf\* ff'\t PCq/TTC /n'S l4 00 Wu ulu 4YLY r vruu.
in pure form by eluting the beads with linker cleavage buffer (in the examples above, either pH F 11 or NaI0 4 -containing buffer), while reacted and unreacted reagents remain bound to the beads.
[00187] Integrating the recently expanded repertoire of synthetic reactions compatible with Q DNA-templated synthesis and the linker strategies described above, multi-step DNA-templated small molecule syntheses can be conducted.
1[00188] In one embodiment, a solution phase DNA-templated synthesis of a non-natural peptide library is described generally below and is shown generally in Figure 35. As shown in SFigure 35, to generate the initial template pool for the library, thirty synthetic biotinylated OC amino oligonucleotides of the sequence format shown in Figure 35 are acylated with one of Sthirty different natural or unnatural amino acids using standard EDC coupling procedures. Four C bases representing a "codon" within each amino acylated primer are designated the identity of the side chain (R 1 The "genetic code" for these side chains are protected with acid labile protecting groups similar to those commonly used in peptide synthesis. The resulting thirty amino acylated DNA primers are annealed to a template DNA oligonucleotide library generated by automated DNA synthesis. Primer extension with a DNA polymerase followed by strand denaturation and purification with streptavidin-linked magnetic beads yield the starting template library (see, Figure 35). As but one general example, a solution phase DNA-templated synthesis of a non-natural peptide library is depicted in Figure 36. The template library is subjected to three DNA-templated peptide bond formation reactions using amino acid reagents attached to decoding DNA oligonucleotides through the sulfone linker described above. Products of each step are purified by preparative denaturing polyacrylamide gel electrophoresis prior to linker cleavage if desired, although this may not be necessary. Each DNA-linked reagent can be synthesized by coupling a 3'-amino terminated DNA oligonucleotide to the encoded amino acid through the bis-NHS carbonate derivative of the sulfone linker as shown in Figure 37. Codons are again chosen so that related codons encode chemically similar amino acids. Following each peptide bond formation reaction, acetic anhydride is used to cap unreacted starting materials and pH 11 buffer is used to effect linker cleavage to expose a new amino group for the next peptide bond formation reaction. Once the tetrapeptide is completed, those library members bearing carboxylate side chains can also be cyclized with their amino termini to form macrocyclic peptides, while linear peptide members can simply be N-acetylated (see Figure 36).
58 of 88 00 WO 02/074929 PCT/US02/08546
O
C1 [00189] It will be appreciated that a virtually unlimited assortment of amino acid building blocks can be incorporated into a non-natural peptide library. Unlike peptide libraries generated using the protein biosynthetic machinery such as phage displayed libraries (O'Neil et al. Curr.
0 Opin. Struct. Biol. 1995, 5, 443-9), mRNA displayed libraries (Roberts et al. Proc. Natl. Acad.
Sci, USA 1997, 94, 12297-12302) ribosome displayed libraries (Roberts et al. Curr. Opin. Chem.
r" Biol. 1999, 3, 268-73; Schaffitzel et al. J. Immunol Methods 1999, 231, 119-35), or intracellular 0 peptide libraries (Norman et al. Science 1999, 285, 591-5), amino acids with non-proteinogenic Sside chains, non-natural side chain stereochemistry, or non-peptidic backbones can all be 00 incorporated into this library. In addition, the many commercially available di-, tri- and oligopeptides can also be used as building blocks to generate longer library members. The presence of non-natural peptides in this library may confer enhanced pharmacological properties such as protease resistance compared with peptides generated ribosomally. Similarly, the macrocyclic library members may yield higher affinity ligands since the entropy loss upon binding their targets may be less than their more flexible linear counterparts. Based on the enormous variety of commercially available amino acids fitting these descriptions, the maximum diversity of this non-natural cyclic and linear tetrapeptde library can exceed 100 x 100 x 100 x 100 10 members.
[00190] Another example of a library using the approach described above includes the DNAtemplated synthesis of a diversity-oriented macrobicyclic library containing 5- and 14-membered rings (Figure 38). Starting material for this library consists of DNA templates aminoacylated with a variety of side-chain protected lysine derivatives and commercially available lysine analogs (including aminoethyl-cysteine, aminoethylserine, and 4-hydroxylysine). In the first step, DNA-templated amide bond formation with a variety of DNA-linked amino acids takes place as described in the non-natural peptide library, except that the vicinal diol linker 16 described above is used instead of the traceless sulfone linker. Following amide bond formation, the diol linker is oxidatively cleaved with sodium periodate. Deprotection of the lysine analog'side chain amine is followed by DNA-templated amide bond formation catalyzed by silver trifluoroacetate between the free amine and a library of acrylic derived thioesters. The resulting olefins are treated with a hydroxylamine to fonn nitrones, which undergo 1,3-dipolar cycloaddition to yield the bicyclic library (Figure 38). DNA-linked reagents for this library are prepared by coupling lysine analogs to 5'-amino-terminated template primers (Figure 59 of 88 00 coupling aminoacylated diol linkers to 3'-amino-terminated DNA oligonucleotides (Figure 38), and coupling acrylic acids to 3'-thiol terminated DNA oligonucleotides (Figure 38).
C [00191] As but one example of a specific library generated from the first general approach described above, three iterated cycles of DNA-templated amide formation, traceless linker cleavage, and purification with streptavidin-linked beads were used to generate a non-natural tripeptide (Figs. 39A and 39B). Each amino acid reagent was linked to a unique biotinylated base DNA oligonucleotide through the sulfone linker described above. The 30-base amine- N terminated template programmed to direct the tripeptide synthesis contained three consecutive regions that were complementary to the three reagents, mimicking the strategy that C, would be used in a multi-step DNA-templated small molecule library synthesis. The first amino acid reagent (13) was combined with the template and activator 4-(4,6-dimethoxy-l,3,5-triazin- 2-yl)-4-methylmorpholinium chloride (DMT-MM) (Kunishima et al. Tetrahedron 2001, 57, 1551) to effect DNA-templated peptide bond formation. The desired product was purified by mixing the crude reaction with streptavidin-linked magnetic beads, washing with 4 M guanidinium chloride, and eluting with pH 11 buffer to effect sulfone linker cleavage, providing product 14. The free amine group in 14 was then elaborated in a second and third round of DNA-templated amide formation and linker cleavage to afford dipeptide 15 and tripeptide 16 (Figures 39A and 39B).
[00192] The progress of each reaction, purification, and sulfone linker cleavage step was followed by denaturing polyacrylamide gel electrophoresis. The final tripeptide linked to template (16) was digested with the restriction endonuclease EcoRI and the digestion fragment containing the tripeptide was characterized by MALDI mass spectrometry. Beginning with 2 nmol 20 fig) of starting material, sufficient tripeptide product was generated to serve as the template for more than 106 in vitro selections and PCR reactions (Kramer et al. in Current Protocols in Molecular Biology, Vol 3 F. M. Ausubel), Wiley, 1999, pp. 15.1) (assuming 1/10,000 molecules survive selection). No significant product was generated when the starting material template was capped with acetic anhydride, or when control reagents containing sequence mismatches were used instead of the complementary reagents (Fig. 39B).
100193] A non-peptidic multi-step DNA-templated small molecule synthesis (Figs. 40A and that uses all three linker strategies developed above was also performed. An amineterminated 30-base template was subjected to DNA-templated amide bond formation using an aminoacyl donor reagent (17) containing the diol linker and a biotinylated oligonucleotide to afford amide of 89 00
O
S 18. The desired product was isolated by capturing the crude reaction on streptavidin beads c followed by cleaving the linker with NaIO 4 to generate aldehyde 19. The DNA-templated Wittig S reaction of 19 with the biotinylated autocleaving phosphorane reagent 20 afforded fumaramide 21. The products from the second DNA-templated reaction were partially purified by washing with streptavidin beads to remove reacted and unreacted reagent. In the third DNA-templated step, fumaramide 21 was subjected to a DNA-templated conjugate addition (Gartner et al. J. Am.
S Chem. Soc. 2001, 123, 6961) using thiol reagent 22 linked through the sulfone linker to a S biotinylated oligonucleotide. The desired conjugate addition product (23) was purified by 00 immobilization with streptavidin beads. Linker cleavage with pH 11 buffer afforded final S product 24 in 5-10% overall isolated yield for the three bond forming reactions, two linker cleavage steps, and three purifications (Figures 40A and 40B). This final product was digested with EcoRI and the mass of the small molecule-linked template fragment was confirmed by MALDI mass spectrometry (exact mass: 2568, observed mass: 2566±5). As in the tripeptide example, each of the three reagents used during this multi-step synthesis annealed at a unique location on the DNA template, and control reactions with sequence mismatches yielded no product (Fig. 40B). As expected, control reactions in which the Wittig reagent was omitted (step 2) also did not generate product following the third step. Taken together, the DNA-templated syntheses of 16 and 24 demonstrate the ability of DNA to direct the sequence-programmed multi-step synthesis of both oligomeric and non-oligomeric small molecules unrelated in structure to nucleic acids.
[00194] The commercial availability of many substrates for DNA-templated reactions including amines, carboxylic acids, a-halo carbonyl compounds, olefins, alkoxyamines, aldehydes, and nitroalkanes may allow the translation of large libraries of DNA into diverse small molecule libraries. The direct one-pot selection of these libraries for members with desired binding or catalytic activities, followed by the PCR amplification and diversification of the DNA encoding active molecules, may enable synthetic small molecules to evolve in a manner paralleling the powerful methods Nature uses to generate new molecular function. In addition, multi-step nucleic acid-templated synthesis is a requirement of previously proposed models I.
Scott, Tetrahedron Lett. 1997, 38, 4961; Li et al. Nature 1994, 369, 218; Tamura et al. Proc.
Natl. Acad. Sci USA 2001, 98, 1393) for the prebiotic translation of replicable information into functional molecules. These findings demonstrate that nucleic acid templates are indeed capable of directing iterative or non-iterative multi-step small molecule synthesis even when reagents 61 of 88 WO 02/074929 PCT/US02/08546 00 O anneal at widely varying distances from the growing molecule (in the above examples, zero to Stwenty bases). As described in more detail below, libraries of synthetic molecules can then be evolved towards active ligand and catalysts through cycles of translation, selection, amplification CM and mutagenesis.
[00195] E) Evolving Plastics: In yet another embodiment of the present invention, a nucleic acid DNA, RNA, derivative thereof) is attached to a polymerization catalyst. Since nucleic C,1 acids can fold into complex structures, the nucleic acid can be used to direct and/or affect the Spolymerization of a growing polymer chain.. For example, the nucleic acid may influence the C,1 selection of monomer units to be polymerized as well as how the polymerization reaction takes Splace stereochemistry, tacticity, activity). The synthesized polymers may be selected for specific properties such molecular, weight, density, hydrophobicity, tacticity, stereoselectivity, etc., and the nucleic acid which formed an integral part of the catalyst which directed its synthesis may be amplified and evolved (Figure 41). Iterated cycles of ligand diversification, selection, and amplification allow for the true evolution of catalysts and polymers towards desired properties.
[00196] To give but one example, a library of DNA molecules is attached to Grubbs' ruthenium-based ring opening metathesis polymerization (ROMP) catalyst through a dihydroimidazole ligand (Scholl et al. Org. Lett. 1(6):953, 1999; incorporated herein by reference) creating a large, diverse pool of potential catalytic molecules, each unique by nature of the functionalized ligand. Undoubtedly, functionalizing the catalyst with a relatively large DNA-dehydroimidazole (DNA-DHI) ligand will alter the activity of the catalyst. Each DNA molecule has the potential to fold into a unique stereoelectronic shape which potentially. has different selectivities and/or activities in the polymerization reaction (Figure 42). Therefore, the library of DNA ligands can be "translated" into a library of plastics upon the addition of various monomers. In certain embodiments, DNA-DHI ligands capable of covalently inserting themselves into the growing polymer, thus creating a polymer tagged with the DNA that encoded its creation, are used. Using the synthetic scheme shown in Figure 42, DHI ligands are produced containing two chemical handles, one used to attach the DNA to the ligand, the other used to attach a pedant olefin to the DHI backbone. Rates of metathesis are known to vary widely based upon olefin substitution as well as the identity of the catalyst. Through alteration of these variable, the rate of pendant olefin incorporation can be modulated such that kpendant olefin metathesis 62 of 88 WO 02/074929 PCTUS02/08546 00 O kROMP, thereby, allowing polymers of moderate to high molecular weights to be formed before insertion of the DNA tag and corresponding polymer termination. Vinylic either are c, commonly used in ROMP to functionalize the polymer termini (Gordon et al. Chem. Biol. 7:9mC 16, 2000; incorporated herein by reference), as well as produce polymers of decreased molecular weight.
[00197] Subsequent selection of a polymer from the library based on a desired property by electrophoresis, gel filtration, centrifugal sedimentation, partitioning into solvents of different Shydrophobicities, etc. Amplification and diversification of the coding nucleic acid via C1 techniques such as error-prone PCR or DNA shuffling followed by attachment to a DHI Sbackbone will allow for production of another pool of potential ROMP catalysts enriched in the S selected activity (Figure 43). This method provides a new approach to generating polymeric materials and the catalysts that create them.
[00198] Example 6: Characterization of DNA-Templated Synthetic Small Molecule Libraries: The non-natural peptide and bicyclic libraries described above are characterized in several stages. Each candidate reagent is conjugated to its decoding DNA oligonucleotide, then subjected to model reactions with matched and mismatched templates. The products from these reactions are analyzed by denaturing polyacrylamide gel electrophoresis to assess reaction efficiency, and by mass spectrometry to verify anticipated product structures. Once a complete set of robust reagents are identified, the complete multi-step DNA-templated syntheses of representative single library members on a large scale is performed and the final products are characterized by mass spectrometry.
(00199] More specifically, the sequence fidelity of each multi-step DNA-templated library synthesis is tested by following the fate of single chemically labeled reagents through the course of one-pot library synthesis reactions. For example, products arising from building blocks bearing a ketone group are captured with commercially available hydrazide-linked resin and analyzed by DNA sequencing to verify sequence fidelity during DNA-templated synthesis.
Similarly, when using non-biotinylated model templates, building blocks bearing biotin groups are purified after DNA-templated synthesis using streptavidin magnetic beads and subjected to DNA sequencing (Liu et al J. Am. Chem. Soc. 2001, 123, 6961-6963) Codons that show a greater propensity to anneal with mismatched DNA are identified by screening in this manner and removed from the genetic code of these synthetic libraries.
63 of 88 WO 02/074929 PCT/US02/08546 00 0 O [00200] Example 7: In Vitro Selection of Protein Ligands from Evolvable Synthetic SLibraries: Because every library member generated in this approach is covalently linked to a SN DNA oligonucleotide that encodes and directs its synthesis, libraries can be subjected to true in
C
vitro selections. Although direct selections for small molecule catalysts of bond-forming or bond-cleaving reactions are an exciting potential application of this approach, the simplest in vitro selection that can be used to evolve these libraries is a selection for binding to a target Sprotein. An ideal initial target protein for the synthetic library selection both plays an important 0 biological role and possesses known ligands of varying affinities for validating the selection C1 methods.
00 S[00201] One receptor of special interest for use in the present invention is the. av33 receptor.
C1 The cavP3 receptor is a member of the integrin family of transmembrane heterodimeric glycoprotein receptors (Miller et al. Drug Discov Today 2000, 5, 397-408; Berman et al. Membr Cell Biol. 2000, 13, 207-44) The av33 integrin receptor is expressed on the surface of many cell types such as osteoclasts, vascular smooth muscle cells, endothelial cells, and some tumor cells.
This receptor mediates several important biological processes including adhesion of osteoclasts to the bone matrix (van der Pluijm et al. J. Bone Miner. Res. 1994, 9, 1021-8) smooth muscle cell migration (Choi et al. J Vasc. Surg. 1994, 19, 125-34) and tumor-induced angiogenesis (Brooks et al. Cell 1994, 79, 1157-64) (the outgrowth of new blood vessels). During tumorinduced angiogenesis, invasive endothelial cells bind to extracellular matrix components through their CXvp3 integrin receptors. Several studies (Brooks et al. Cell 1994, 79, 1157-64; Brooks et al. Cell 1998, 92, 391-400; Friedlander et al. Science 1995, 270, 1500-2; Varner et al. Cell Adhes Commun 1995, 3, 367-74; Brooks et al. J. Clin Invest 1995, 96, 1815-22) have demonstrated that the inhibition of this integrin binding event with antibodies or small synthetic peptides induces apoptosis of the proliferative angiogenic vascular cells and can inhibit tumor metastasis.
[00202] A number of peptide ligands of varying affinities and selectivities for the CLP33 integrin receptor have been reported. Two benchmark avp3 integrin antagonists are the linear peptide GRGDSPK (ICso 210 nM (Dechantsreiter et al. J Med. Chem. 1999, 42, 3033-40; Pfaff et al. J Biol. Chem. 1994, 269, 20233-8) and the cyclic peptide cyclo-RGDfV (Pfaff et al.
J. Biol. Chem. 1994, 269, 20233-8)'(f= (D)-Phe, ICso 10 nM). While peptides antagonists for integrins commonly contain RGD, not all RGD-containing peptides are high affinity integrin ligands. Rather, the conformational context of RGD and other peptide sequences can have a 64 of 88 WO 02/074929 PCT/US02/08546 00
O
S profound effect on integrin affinity and specificity (Wermuth et al. J. Am. Chem. Soc. 1997, 119, S1328-1335; Geyer et a. J. Am. Chem. Soc. 1994, 116, 7735-7743; Rai et al. Bioorg. Med. Chem.
c- Lett 2001, 11, 1797-800; Rai et al. Curr. Med. Chem. 2001, 8, 101-19) For this reason, Q combinatorial approaches towards avp3 integrin receptor antagonist discovery are especially promising.
r> [00203] The biologically important and medicinally relevant role of the avP3 integrin receptor 0 together with its known peptide antagonists and its commercial availability (Chemicon 0 International, Inc., Temecula, CA) make the tvP3 integrin receptor an ideal initial target for 00 DNA-templated synthetic small molecule libraries. The avP3 integrin receptor can be Simmobilized by adsorption onto microtiter plate wells without impairing its ligand binding ability or specificity (Dechantsreiter et al. J. Med. Chem. 1999, 42, 3033-40; Wermuth et al. J.
Am. Chem. Soc. 1997, 119, 1328-1335; Haubner et al. J. Am. Chem. Soc. 1996, 118, 7461-7472).
Alternatively, the receptor can be immobilized by conjugation with NHS ester or maleimide groups covalently linked to sepharose beads and the ability of the resulting integrin affinity resin to maintain known ligand binding properties can be verified.
[00204] To perform the actual protein binding selections, DNA template-linked synthetic peptide or macrocyclic libraries are dissolved in aqueous binding buffer in one pot and equilibrated in the presence of immobilized avP 3 integrin receptor. Non-binders are washed away with buffer. Those molecules that may be binding through their attached DNA templates rather than through their synthetic moieties are eliminated by washing the bound library with unfunctionalized DNA templates lacking PCR primer binding sites. Remaining ligands bound to the immobilized avP3 integrin receptor are eluted by denaturation or by the addition of excess high affinity RGD-containing peptide ligand. The DNA templates that encode and direct the syntheses of avP3 integrin binders are amplified by PCR using one primer designed to bind to a constant 3' region of the template and one pool of biotinylated primers functionalized at its end with the library starting materials (Fig. 44). Purification of the biotinylated strand completes one cycle of synthetic molecule translation, selection, and amplification, yielding a subpopulation of DNA templates enriched in sequences that encode synthetic avP3 integrin ligands.
[00205] For reasons similar to those that make the cxVP3 integrin receptor an attractive initial target for the approach to generating synthetic molecules with desired properties, the factor Xa serine protease also serves as a promising protein target. Blood coagulation involves a complex of 88 WO 02/074929 PCT/US02/08546 00
O
O cascade of enzyme-catalyzed reactions that ultimately generate fibrin, the basis of blood clots (Rai et al. Curr. Med Chem. 2001, 8, 101-109; Vacca et al. Curr. Opin. Chem Biol. 2000, 4, 394-400) Thrombin is the serine protease that converts fibrinogen into fibrin during blood C clotting. Thrombin, in turn, is generated by the proteolytic action of factor Xa on prothrombin.
Because thromboembolitic (blood clotting) diseases such as stroke remain a leading cause of death in the world (Vacca et al. Curr. Opin. Chem. Biol. 2000, 4, 394-400) the development of CN drugs that inhibit thrombin or factor Xa is a major area of pharmaceutical research. The Sinhibition of factor Xa is a newer approach thought to avoid the side effects associated with N inhibiting thrombin, which is also involved in normal hemostasis (Maignan et al. J. Med. Chem.
00 S2000, 43, 3226-32; Leadley et al. J. Cardiovasc. Pharmacol. 1999, 34, 791-9; Becker et al.
SBioorg. Med. Chem. Lett. 1999, 9, 2753-8; Choi-Sledesld et al. Bioorg. Med. Chem. Lett. 1999, 9, 2539-44; Choi-Sledeski et al. J. Med. Chem. 1999, 42, 3572-87; Ewing et al. J. Med. Chem.
1999, 42, 3557-71; Bostwick et al. Thromb Haemost 1999, 81, 157-60). Although many agents including heparin, hirudin, and hirulog have been developed to control the production of thrombin, these agents generally have the disadvantage of requiring intravenous or subcutaneous injection several times a day in addition to possible side effects, and the search for synthetic small molecule factor Xa inhibitors remains the subject of great research effort.
[00206] Among factor Xa inhibitors with known binding affinities are a series of tripeptides ending with arginine aldehyde (Marlowe et al. Bioorg. Med. Chem. Lett. 2000, 10, 13-16) that are easily be included in the DNA-templated non-natural peptide library described above.
Depending on the identities of the first two residues, these tripeptides exhibit IC 5 0 values ranging from 15 nM to 60 uM (Marlowe et al. Bioorg. Med. Chem. Lett. 2000, 10, 13-16) and therefore provide ideal positive controls for validating and calibrating an in vitro selection for synthetic factor Xa ligands (see below). Both factor Xa and active factor Xa immobilized on resin are commercially available (Protein Engineering Technologies, Denmark). The resin-bound factor Xa is used to select members of both the DNA-templated non-natural peptide and bicyclic libraries with factor Xa affinity in a manner analogous to the integriri receptor binding selections described above.
[00207] Following PCR amplification of DNA templates encoding selected synthetic molecules, additional rounds of translation, selection, and amplification are conducted to enrich the library for the highest affinity binders. The stringency of the selection is gradually increased 66 of 88 WO 02/074929 PCT/US02/08546 00 0 O by increasing the salt concentration of the binding and washing buffers, decreasing the duration Sof binding, elevating the binding and washing temperatures, and increasing the concentration of washing additives such as template DNA or unrelated proteins. Importantly, in vitro selections CC can also select for specificity in addition to binding affinity. To eliminate those molecules that possess undesired binding properties, library members bound to immobilized cvP33 integrin or factor Xa are washed with non-target proteins such as other integrins or other serine proteases, C leaving only those molecules that bind the target protein but do not bind non-target proteins.
O 00208] Iterated cycles of translation, selection, and amplification results in library enrichment 00 SDiversification of these synthetic libraries are achieved in at least two ways, both analogous to r, methods used by Nature to diversify proteins. Random point mutagenesis is performed by conducting the PCR amplification step under error-prone PCR (Caldwell et al. PCR Methods Applic. 1992, 2, 28-33) conditions. Because the genetic code of these molecules are written to assign related codons to related chemical groups, similar to the way that the natural protein genetic code is constructed, random point mutations in the templates encoding selected molecules will diversify progeny towards chemically related analogs. In addition to point mutagenesis, synthetic libraries generated in this approach are also diversified using recombination. Templates to be recombined have the structure shown in Fig. 45, in which codons are separated by five-base non-palindromic restriction endonuclease cleavage sites such as those cleaved by Avail (G/GWCC, W=A or Sau96I (G/GNCC, N=A, G, T, or DdeI (C/TNAG), or HinFI (G/ANTC). Following selections, templates encoding desired molecules are enzymatically digested with these commercially available restriction enzymes. The digested fragments are then recombined into intact templates with T4 DNA ligase. Because the restriction sites separating codons are nonpalindromic, templates fragments can only reassemble to form intact recombined templates (Fig. 45). DNA-templated translation of recombined templates provides recombined small molecules. In this way, functional groups between synthetic small molecules with desired activities are recombined in a manner analogous to the recombination of amino acid residues between proteins in Nature. It is well appreciated that recombination explores the sequence space of a molecule much more efficiently than point mutagenesis alone (Minshull et al. Curr. Opin. Chem. Biol. 1999, 3, 284-90; Bogarad et al. Proc.
Natl. Acad. Sci. USA 1999, 96, 2591-5; Stemmer, W. Nature 1994, 370, 389-391).
67 of 88 WO 02/074929 PCT/US02/08546 00 [00209] Small molecule evolution using mutation and recombination offers two potential advantages over simple enrichment. If the total diversity of the library is much less than the number of molecules made (typically 1012 to 1015), every possible library member is present at C< the start of the selection. In this case, diversification is still useful because selection conditions almost always change as rounds of evolution progress. For example, later rounds of selection will likely be conducted under higher stringencies, and may involve counter selections against C1 binding non-target proteins. Diversification gives library members that have been discarded during earlier rounds of selection the chance to reappear in later rounds under altered selection ,1 conditions in which their fitness relative to other members may be greater. In addition, it is quite Spossible using this approach to generate a synthetic library that has a theoretical diversity greater than 1015 molecules. In this case, diversification allows molecules that never existed in the original library to emerge in later rounds of selections on the basis of their similarity to selected molecules, similar to the way in. which protein evolution searches the vastness of protein sequence space one small subset at a time.
[002101 Example 8: Characterization of Evolved Compounds: Following multiple rounds of selection, amplification, diversification, and translation, molecules surviving the selection will be characterized for their ability to bind the target protein. To identify the DNA sequences encoding evolved synthetic molecules surviving the selection, PCR-amplified templates are cloned into vectors, transformed into cells, and sequenced as individual clones. DNA sequencing of these subcloned templates reveal the identity of the synthetic molecules surviving the selection. To gain general information about the functional groups being selected during rounds of evolution, populations of templates are sequenced in pools to reveal the distribution of A, G, T, and C at every codon position. The judicious design of each functional group's genetic code allows considerable information to be gathered from population sequencing. For example, a G at the first position of a codon may designate a charged group, while a C at this position may encode a hydrophobic substituent.
[00211] To validate the integrin binding selection and to compare selected library members with known CvP3 3 integrin ligands, linear GRGDSPK and a cyclic RGDfV analog (cyclic iso- ERGDfV) are also included in the DNA-templated cyclic peptide library. The selection conditions are adjusted until verification that libraries containing these known integrin ligands undergo enrichment of the DNA templates encoding the known ligands upon selection for 68 of 88 WO 02/074929 PCT/US02/08546 00 0 O integrin binding. In addition, the degree of enrichment of template sequences encoding these known ovP3 integrin ligands is correlated with their known affinities and with the enrichment C. and affinity of newly discovered avP3 integrin ligands.
M
n [00212] Once the enrichment of template sequences encoding known and new integrin ligands is confirmed, novel evolved ligands will be synthesized by non-DNA templated synthesis and assayed for their avP 3 integrin receptor antagonist activity and specificity. Standard in vitro binding assays to integrin receptors (Dechantsreiteret al. Med. Chem. 1999, 42, 3033-40) are Sperformed by competing the binding of biotinylated fibrinogen (a natural integrin ligand) to immobilized integrin receptor with the ligand to be assayed. The inhibition of binding to 00 O fibrinogen is quantitated by incubation with an alkaline phosphatase-conjugated anti-biotin (1 antibody and a chromogenic alkaline phosphate substrate. Comparison of the binding affinities of randomly chosen library members before and after selection will validate the evolution of the library towards target binding. Assays for binding non-target proteins reveal the ability of these libraries to be evolved towards binding specificity in addition to binding affinity.
[00213] Similarly, the selection for factor Xa binding is validated by including the known factor Xa tripeptide inhibitors in the library design and verifying that a round of factor Xa binding selection and PCR amplification results in the enrichment of their associated DNA templates. Synthetic library members evolved to bind factor Xa are assayed in vitro for their ability to inhibit factor Xa activity. Factor Xa inhibition can be readily assayed spectrophotometrically using the commercially available chromogenic substrate S-2765 (Chromogenix, Italy).
[00214] While the DNA sequence alone of a non-natural peptide library member is likely to reveal the exact identity of the corresponding peptide, the final step in the bicyclic library synthesis is a non-DNA-templated intramolecular 1,3-dipolar cycloaddition that may yield diastereomeric pairs of regioisomers. Although modeling strongly suggests that only the regioisomer shown in Fig. 38 can form for steric reasons, facial selectivity is less certain.
Diastereomeric purity is not a requirement for the in vitro selections described above since each molecule is selected on a single molecule basis. Nevertheless, it may be useful to characterize the diastereoselectivity of the dipolar cycloaddition. To accomplish this, non-DNA-templated synthesis of selected bicyclic library members is performed, diastereomers are separated by chiral preparative HPLC, and product stereochemistry by nOe or X-ray diffraction is determined.
69 of 88 WO 02/074929 PCT/US02/08546 00 O [00215] Example 9: Translating DNA into Non-Natural Polymers Using DNA SPolymerases: An alternative approach to translating DNA into non-natural, evolvable polymers takes advantage of the ability of some DNA polymerases to accept certain modified nucleotide C triphosphate substrates M. Perrin et al. J. Am. Chem. Soc. 2001, 123, 1556; D. M. Perrin et al. Nucleosides Nucleotides 1999, 18, 377-91; T. Gourlain et al. Nucleic Acids Res. 2001, 29, 1898-1905; S. E. Lee et al. Nucleic Acids Res. 2001, 29, 1565-73; K. Sakthievel et al. Angew.
Cr Chem. Int. Ed. 1998, 37, 2872-2875). Several deoxyribonucleotides (Fig. 45) and ribonucleotides Sbearing modifications to groups that do not participate in Watson-Crick hydrogen bonding are C known to be inserted with high sequence fidelity opposite natural DNA templates. Importantly, Ssingle-stranded DNA containing modified nucleotides can serve as efficient templates for the SDNA-polymerase-catalyzed incorporation of natural or modified mononucleotides. In one of the earliest examples of modified nucleotide incorporation by DNA polymerase, Toole and coworkers reported the acceptance of 5-(l-pentynyl)-deoxyuridine 1 by Vent DNA polymerase under PCR conditions A. Latham et al. Nucleic Acids Res. 1994, 22, 2817-22). Several additional 5-functionalized deoxyuridines derivatives were subsequently found to be accepted by thermostable DNA polymerases suitable for PCR Sakthievel et al. Angew.
Chem. Int. Ed. 1998, 37, 2872-2875). The first functionalized purine accepted by DNA polymerase, deoxyadenosine analog 8, was incorporated into DNA by T7 DNA polymerase together with deoxyuridine analog 7 M. Perrin et al. Nucleosides Nucleotides 1999, 18, 377- 91). DNA libraries containing both 7 and 8 were successfully selected for metal-independent RNA cleaving activity M. Perrin et al. J. Am. Chem. Soc. 2001, 123, 1556-63). Williams and co-workers recently tested several deoxyuridine derivatives for acceptance by Taq DNA polymerases and concluded that acceptance is greatest when using CS-modified uridines bearing rigid alkyne or trans-alkene groups such as 9 and 10 E. Lee et al. Nucleic Acids Res. 2001, 29, 1565-73). A similar study Gourlain et al. Nucleic Acids Res. 2001, 29, 1898-1905) on C7-functionalized 7-deaza-deoxyadenosines revealed acceptance by Taq DNA polymerase of 7aminopropyl- cis-7-aminopropenyl- and 7-aminopropynyl-7-deazadeoxyadenosine (13).
[00216] The functionalized nucleotides incorporated by DNA polymerases to date, shown in Fig. 46, have focused on adding "protein-like" acidic and basic functionality to DNA. While equipping nucleic acids with general acid and general base functionality such as primary amine of 88 WO 02/074929 PCT/US02/08546 00 0 O and carboxylate groups may increase the capability of nucleic acid catalysts, the functional Sgroups present in natural nucleic acid bases already have demonstrated the ability to serve as n general acids and bases. The hepatitis delta ribozyme, for example, is thought to use the pKa- MC modulated endocyclic amine of cytosine 75 as a general acid Nakano et al. Science 2000, 287, 1493-7) and the peptidyl transferase activity of the ribosome may similarly rely on general base or general acid catalysis W. Muth et al. Science 2000, 289, 947-50; P. Nissen et al.
C Science 2000, 289, 920-930; N. Ban et al. Science 2000, 289, 905-920) although the latter case 0 remains the subject of ongoing debate Polacek et al. Nature 2001, 411, 498-501). Equipping CN DNA bases with additional Bronsted acidic and basic groups, therefore, may not profoundly 00 0expand the scope of DNA catalysis.
p [00217] In contrast with simple general acid and general base functionality, chiral metal centers would expand considerably the chemical scope of nucleic acids. Functionality aimed at binding chemically potent metal centers has yet to been incorporated into nucleic acid polymers.
Natural DNA has demonstrated the ability to fold in complex three-dimensional structures capable of stereospecifically binding target molecules H. Lin et al. Chem. Biol. 1997, 4, 817- 32; C. H. Lin et al. Chem. Biol. 1998, 5, 555-72; P. Schultze et al. J. Mol. Biol. 1994, 235, 1532- 47) or catalyzing phosphodiester bond manipulation W. Santoro et al. Proc. Natl. Acad. Sci.
USA 1997, 94, 4262-6; R. R. Breaker et al. Chem. Biol. 1995, 2, 655-60; Y. Li et al.
Biochemistry 2000, 39, 3106-14; Y. Li et al. Proc. Natl. Acad. Sci. USA 1999, 96, 2746-51).
DNA depurination L. Sheppard et al. Proc. Natl. Acad. Sci. USA 2000; 97, 7802-7807) and porphyrin metallation Li et al. Biochemistry 1997, 36, 5589-99; Y. Li et al. Nat. Struct. Biol.
1996, 3, 743-7). Non-natural nucleic acids augmented with the ability to bind chemically potent, water-compatible metals such Cu, La, Ni, Pd, Rh, Ru, or Sc may possess greatly expanded catalytic properties. For example, a Pd-binding oligonucleotide folded into a well-defined structure may possess the ability to catalyze Pd-mediated coupling reactions with a high degree of regiospecificity or stereospecificity. Similarly, non-natural nucleic acids that form chiral Sc binding sites may serve as enantioselective cycloaddition or aldol addition catalysts. The ability of DNA polymerases to translate DNA sequences into these non-natural polymers coupled with in vitro selections for catalytic activities would therefore enable the direct evolution of desired catalysts from random libraries.
71 of 88 WO 02/074929 PCT/US02/08546 00 o O [00218] Evolving catalysts in this approach addresses the difficulty of rationally designing catalytic active sites with specific chemical properties that has inspired recent combinatorial approaches W. Kuntz et al. Curr. Opin. Chem. Biol. 1999, 3, 313-319; M. B. Francis et al.
c Curr. Opin. Chem. Biol. 1998, 2, 422-8) to organometallic catalyst discovery. For example, Hoveyda and co-workers identified Ti-based enantioselective epoxidation catalysts by serial screening of peptide ligands D. Shimizu et al. Angew. Chem. Int. Ed. 1997, 36) Serial CN screening was also used by Jacobsen and co-workers to identify peptide ligands that form 0enantioselective epoxidation catalysts when complexed with metal cations B. Francis et al.
(K Angew. Chem. Int. Ed. Engl. 1999, 38, 937-941) Recently, a peptide library containing Sphosphine side chains was screened for the ability to catalyze malonate ester addition to Scyclopentenyl acetate in the presence of Pd R. Gilbertson et al. J. Am. Chem. Soc. 2000, 122, 6522-6523). The current approach differs fundamentally from previous combinatorial catalyst discovery efforts, however, in that it enables catalysts with desired properties to spontaneously emerge from one pot, solution-phase libraries after evolutionary cycles of diversification, amplification, translation, and selection. This strategy allows up to 1015 different catalysts to be generated and selected for desired properties in a single experiment. The compatibility of our approach with one-pot in vitro selections allows the direct selection for reaction catalysis rather than screening for a phenomenon associated with catalysis such as metal binding or heat generation. In addition, properties difficult to screen rapidly such as substrate stereospecificity or metal selectivity can be directly selected using our approach (see below).
[00219] Key intermediates for a number of C5-functionalized uridine analogs and C7ftuctionalized 7-deazaadenosine analogs have been synthesized for incorporation into nonnatural DNA polymers. In addition, the synthesis of six C8-functionalized adenosine analogs as deoxyribonucleotide triphosphates has been completed. Because only limited information exists on the ability of DNA polymerases to accept modified nucleotides, we chose to synthesize analogs were synthesized that not only will bring metal-binding functionality to nucleic acids but that also will provide insights into the determinants of DNA polymerase acceptance.
[00220] The strategy for the synthesis of metal-binding uridine and 7-deazaadenosine analogs is shown in Fig. 47. Both routes end with amide bond formation between NHS esters of metalbinding functional groups and amino modified deoxyribonucleotide triphosphates (7 and 13).
Analogs 7 and 13 as well as acetylated derivatives of 7 have been previously shown M.
72 of 88 WO 02/074929 PCT/US02/08546 00 O Perrin et al. J. Am. Chem. Soc. 2001, 123, 1556-63; D. M. Perrin et al. Nucleosides Nucleotides S1999, 18, 377-91; J. A. Latham et al. Nucleic Acids Res. 1994, 22, 2817-22; T. Gourlain et al.
TO Nucleic Acids Res. 2001, 29, 1898-1905; S. E. Lee et al. Nucleic Acids Res. 2001, 29, 1565-73; mC K. Sakthivel et al. Angew. Chem. Int. Ed. Engl. 1998, 37, 2872-2875) to be tolerated by DNA polymerases, including thermostable DNA polymerases suitable for PCR. This convergent approach allows a wide variety of metal-binding ligands to be rapidly incorporated into either 1 nucleotide analog. The synthesis of 7 has been completed following a previously reported (K.
SSakthivel et al. Angew. Chem. Int. Ed. Engl. 1998, 37, 2872-2875) route (Fig. 48, Phillips, C1 Chorba, Liu, unpublished results). Heck coupling of commercially available 5-iodo-2'- 00 Sdeoxyuridine (22) with N-allyltrifluoroacetamide provided 23. The 5'-triphosphate group was Sinstalled by treatment of 23 with trimethylphosphate, POC13, and proton sponge (1,8bis(dimethylamino)-naphthalene) followed by tri-n-butylammonium pyrophosphate, and the trifluoroacetamide group then removed with aqueous ammonia to afford 7.
[00221] Several steps towards the synthesis of 13 have been completed, the key intermediate for 7-deazaadenosine analogs (Fig. 49). Following a known route Davoll. J Am. Chem. Soc.
1960, 82, 131-138) diethoxyethylcyanoacetate (24) was synthesized from bromoacetal 25 and ethyl cyanoacetate Condensation of 24 with thiourea provided pyrimidine 27, which was desulfurized with Raney nickel and then cyclized to pyrrolopyrimidine 28 with dilute aqueous HC1. Treatment of 28 with POC13 afforded 4-chloro-7-deazaadenine The aryl iodide group which will serve as a Sonogashira coupling partner for installation of the propargylic amine in 13 was installed by reacting 29 with N-iodosuccinimide to generate 4-chloro-7-iodo-7-deazaadenine in 13% overall yield from bromoacetal [00222] As alternative functionalized adenine analogs that will both probe the structural requirements of DNA polymerase acceptance and provide potential metal-binding functionality, six 8-modified deoxyadenosine triphosphates (Fig. 50) have been synthesized. All functional groups were installed by addition to 8-bromo-deoxyadenosine which was prepared by bromination of deoxyadenosine in the presence of ScCl 3 which we found to greatly increase product yield. Methyl- ethyl- and vinyladenosine (34) were synthesized by Pdmediated Stille coupling of the corresponding alkyl tin reagent and 31 Mamos et al.
Tetrahedron Lett. 1992, 33, 2413-2416). Methylamino- (35) Nandanan et al. J. Med. Chem.
1999, 42, 1625-1638), ethylamino- and histaminoadenosine (37) were prepared by 73 of 88 WO 02/074929 PCT/US02/08546 00
O
O treatment of 23 with the corresponding amine in water or ethanol. The triphosphates of 32-37 were synthesized as described above.
[002231 The ability of thermostable DNA polymerases suitable for PCR amplification to Caccept these modified nucleotide triphosphates containing metal-binding functionality. Nonnatural nucleotide triphosphates were purified by ion exchange HPLC and added to PCR reactions containing Taq DNA polymerase, three natural deoxynucleotide triphosphates, pUC19 CN template DNA, and two DNA primers. Primers were chosen to generate PCR products ranging 0from 50 to 200 base pairs in length. Control PCR reactions contained the four natural C, deoxynucleotide triphosphates and no non-natural nucleotides. PCR reactions were analyzed by Sagarose or denaturing acrylamide gel electrophoresis. Amino modified uridine derivative 7 was efficiently incorporated by Taq DNA polymerase over 30 PCR cycles, while the triphosphate of 23 was not an efficient polymerase substrate (Fig. 51). Previous findings on the acceptance of 7 by Taq DNA polymerase are in conflict, with both non-acceptance Sakthivel et al. Angew.
Chem. Int. Ed. Engl. 1998, 37, 2872-2875) and acceptance E. Lee et al. Nucleic Acids Res.
2001, 29, 1565-73) reported.
[00224] Non-Natural Metal-Binding Deoxyribonucleotide Triphosphate Synthesis: The syntheses of the C5-functionalized uridine, C7-functionalized 7-deazaadenosine, and C8functionalized adenosine deoxynucleotide triphosphates will be completed. Synthesis of the 7deazaadenosine derivatives from 4-chloro-7-iodo-deazaadenine (30) proceeds by glycosylation of 30 with protected deoxyribosyl chloride 38 followed by ammonolysis to afford 7-iodoadenosine (39) (Fig. 31) (Gourlain et al. Nucleic Acids Res. 2001, 29, 1898-1905). Protected deoxyribosyl chloride 38 can be generated from deoxyribose as shown in Fig. 52. Pd-mediated Sonogashira coupling (Seela et al. Helv. Chem. Acta 1999, 82, 1878-1898) of 39 with Npropynyltrifluoroacetamide provides 40, which is then be converted to the 5' nucleotide triphosphate and deprotected with ammonia as described above to yield 13.
[00225] To generate rapidly a collection of metal-binding uridine and adenosine analogs, a variety of metal-binding groups as NHS esters will be coupled to C5-modified uridine intennediate 7 (already synthesized) and C7-modified 7-deazaadenosine intermediate 13. Metalbinding groups that will be examined initially are shown in Fig. 47 and include phosphines, thiopyridyl groups, and hemi-salen moieties. If our initial polymerase acceptance assays (see the following section) of triphosphates of 8-modified adenosines 32-37 (Fig. 50) suggest that a 74 of 88 WO 02/074929 PCT/US02/08546 00 O variety of 8-modified adenosine analogs are accepted by thermostable polymerases, alkyl- and Svinyl trifluoroacetamides will be coupled to 8-bromo-deoxyadenosine (31) to generate nucleotide triphosphates such as 41 and 42 (Fig. 53). These intermediates are then coupled with the NHS C esters shown in Fig. 46 to generate a variety of metal-binding 8-functionalized deoxyadenosine triphosphates.
[00226] Evaluating Non-Natural Nucleotides: Each functionalized deoxyribonucleotide C1 triphosphate is then assayed for its suitability as a building block of an evolvable non-natural Spolymer library in two stages. First, simple acceptance by thermostable DNA polymerases is C measured by PCR amplification of fragments of DNA plasmid pUC19 of varying length. PCR 00 reactions contain synthetic primers designed to bind at the ends of the fragment, a small quantity Sof pUC19 template DNA, a thermostable DNA polymerase (Taq, Pfu or Vent), three natural deoxyribonucleotide triphosphates, and the non-natural nucleotide triphosphate to be tested. The completely successful incorporation of the non-natural nucleotide results in the production of DNA products of any length at a rate similar to that of the control reaction. Those nucleotides that allow at least incorporation of 10 or more non-natural nucleotides in a single product molecule with at least modest efficiency are subjected to the second stage of evaluation.
[00227] Non-natural nucleotides accepted by thermostable DNA polymerases are evaluated for their possible mutagenic properties. If DNA polymerases insert a non-natural nucleotide opposite an incorrect (non-Watson-Crick) template base, or insert an incorrect natural nucleotide opposite a non-natural nucleotide in the template, the fidelity of library amplification and translation is compromised. To evaluate this possibility, PCR products generated in the above assay are subjected to DNA sequencing using each of the PCR primers. Deviations from the sequence of the pUC19 template imply that one or both of the mutagenic mechanisms are taking place. Error rates of less than 0.7% per base per 30 PCR cycles are acceptable, as error-prone PCR generates errors at approximately this rate (Caldwell et al. PCT Methods Applic. 1992, 2, 28-33) yet has been successfully used to evolve nucleic acid libraries.
[00228] Pairs of promising non-natural adenosine analogs and non-natural uridine analogs are also tested together for their ability to support DNA polymerization in a PCR reaction containing both modified nucleotide triphosphates together with dGTP and dCTP. Successful PCR product formation with two non-natural nucleotide triphosphates enables the incorporation of two nonnatural metal-binding bases into the same polymer molecule. Functionalized nucleotides that are of 88 WO 02/074929 PCT/US02/08546 00
O
C especially interesting yet are not compatible with Taq, Pfu, or Vent thermostable DNA polymerases can still be used in the libraries provided that they are accepted by a commercially Savailable DNA polymerase such as the Klenow fragment of E. coli DNA polymerase I, T7 DNA C polymerase, T4 DNA polymerase, or M-MuLV reverse transcriptase. In this case, the assays require conducting the primer extension step of the PCR reaction at 25-37 0 C, and fresh polymerase must be added at every cycle following the 94°C denaturation step. DNA C1 sequencing to evaluate the possible mutagenic properties of the non-natural nucleotide is still performed as described above S[00229] Generating Libraries of Metal-Binding Polymers: Based on the results of the above Snon-natural nucleotide assays, several libraries of-1015 different nucleic acid sequences will be Smade containing one or two of the most polymerase compatible and chemically promising nonnatural metal-binding nucleotides. Libraries are generated by PCR amplification of a synthetic DNA template library consisting of a random region of 20 or 40 nucleotides flanked by two base constant priming regions (Fig. 54). The priming regions contain restriction endonuclease cleavage sites to allow cloning into vectors for DNA sequencing of pools or individual library members. One primer contains a chemical handle such as a primary amine group or a thiol group at its 5' terminus and becomes the coding strand of the library. The other primer contains a biotinylated T at its 5' terminus and becomes the non-coding strand. The PCR reaction includes one or two non-natural metal-binding deoxyribonucleotide triphosphates, three or two natural deoxyribonucleotide triphosphates, and a DNA polymerase compatible with the nonnatural nucleotide(s). Following PCR reaction to generate the double-stranded form of the library and gel purification to remove unused primers, library member duplexes are denatured chemically. The non-coding strands are the removed by several washings with streptavidinlinked magnetic beads to ensure that no biotinylated strands remain in the library. Libraries of up to 1015 different members are generated by this method, far exceeding the combined diversity of previous combinatorial catalyst efforts.
[00230] Each library is then incubated in aqueous solution with a metal of interest from the following non-limiting list of water compatible metal salts (Fringueli et al. Eur. J. Org. Chem.
2001, 2001, 439-455; Zaitoun et al. J. Phys. Chem. B 1997, 1857-1860): ScCl 3 CrCl 3 MnC1 2 FeC1 2 FeCl 3 CoC12, NiC12, CuC12, ZnCl 2 GaC13, YC13, RuCl 3 RhC13, PdC12, AgC1, CdC1 2 InC1 3 SnC12, La(OTf) 3 Ce(OTf) 3 Pr(OTf) 3 Nd(OTf) 3 Sm(OTf) 3 Eu(OTf) 3 Gd(OTf) 3 76 of 88 WO 02/074929 PCT/US02/08546 00 STb(OTf) 3 Dy(OTf) 3 Ho(OTf) 3 Er(OTf) 3 Tm(OTf) 3 Yb(OTf) 3 Lu(OTf) 3 IrCl 3 PtCl 2 AuC1, HgCl 2 HgCl, PbC12, or BiC1 3 Metals are chosen based on the specific chemical reactions Sto be catalyzed. For example, libraries aimed at reactions such as aldol condensations or hetero Cc Diels-Alder reactions that are known (Fringuelli et al. Eur. J. Org. Chem. 2001, 2001, 439-455) to be catalyzed by Lewis acids are incubated with ScCl 3 or with one of the lanthanide triflates, while those aimed at coupling electron-deficient olefins with aryl halides are incubated with Ci PdC12. The metalated library is then purified away from unbound metal salts by gel filtration 0using sephadex or acrylamide cartridges, which separate DNA oligonucleotides 25 bases or C longer from unbound small molecule components.
0 [00231] The ability of the polymer library (or of individual library members) to bind metals of Sinterest is verified by treating the metalated library free of unbound metals with metal staining reagents such as dithiooxamide, dimethylglyoxime, KSCN (Francis et al. Curr. Opin. Chem.
Biol. 1998, 2, 422-8) or EDTA (Zaitoun et al. J. Phys. Chem. B 1997, 101, 1857-1860) that become distinctly colored in the presence of different metals. The approximate level of metal binding is measured by spectrophotometric comparison with solutions of free metals of known concentration and with solutions of positive control oligonucleotides containing an EDTA group (which can be introduced using a commercially available phosphoramidite from Glen Research).
[00232] In Vitro Selections for Non-Natural Polymer Catalysts: Metalated libraries of evolvable non-natural polymers containing metal-binding groups are then subjected to one-pot, solution-phase selections for catalytic activities of interest. Library members that catalyze virtually any reaction that causes bond formation between two substrate molecules or that results in bond breakage into two product molecules are selected using the schemes proposed in Figs. 54 and 55. To select for bond forming catalysts (for example, hetero Diels-Alder, Heck coupling, aldol reaction, or olefin metathesis catalysts), library members are covalently linked to one substrate through their 5' amino or thiol termini. The other substrate of the reaction is synthesized as a derivative linked to biotin. When dilute solutions of library-substrate conjugate are reacted with the substrate-biotin conjugate, those library members that catalyze bond formation cause the biotin group to become covalently attached to themselves. Active bond forming catalysts can then be separated from inactive library members by capturing the former with irmnobilized streptavidin and washing away inactive polymers (Fig. 77 of 88 00
O
[00233] In an analogous manner, library members that catalyze bond cleavage reactions such as retro-aldol reactions, amide hydrolysis, elimination reactions, or olefin dihydroxylation M followed by periodate cleavage can also be selected. In this case, metalated library members are covalently linked to biotinylated substrates such that the bond breakage reaction causes the disconnection of the biotin moiety from the library members (Fig. 56). Upon incubation under CN reaction conditions, active catalysts, but not inactive library members, induce the loss of their biotin groups. Streptavidin-linked beads can then be used to capture inactive polymers, while C active catalysts are able to elute from the beads. Related bond formation and bond cleavage 00 selections have been used successfully in catalytic RNA and DNA evolution (Jaschke et al. Curr.
CN Opin. Chem. Biol. 2000, 4, 257-62) Although these selections do not explicitly select for multiple turnover catalysis, RNAs and DNAs selected in this manner have in general proven to be multiple turnover catalysts when separated from their substrate moieties (Jischke et al. Curr.
Opin. Chem. Biol. 2000, 4, 257-62; Jaeger et al. Proc. Natl. Acad. Sci. USA 1999, 96, 14712-7; Bartel et al. Science, 1993, 261, 1411-8; Sen et al. Curr. Opin. Chem. Biol. 1998, 2, 680-7).
[00234] Catalysts of three important and diverse bond-forming reactions will initially be evolved: Heck coupling, hetero Diels-Alder cycloaddition, and aldol addition. All three reactions are water compatible (Kobayashi et al. J. Am. Chem. Soc. 1998, 120, 8287-8288; Fringuelli et al. Eur. J. Org. Chem. 2001, 2001, 439-455; Li et al. Organic Reactions in Aqueous Media: Wiley and Sons: New York, 1997) and are known to be catalyzed by metals. As Heck coupling substrates both electron deficient and unactivated olefins will be used together with aryl iodides and aryl chlorides. Heck reactions with aryl chlorides in aqueous solution, as well as room temperature Heck reactions with non-activated aryl chlorides, have not yet been reported to our knowledge. Libraries for Heck coupling catalyst evolution use PdCI 2 as a metal source.
Hetero Diels-Alder substrates include simple dienes and aldehydes, while aldol addition substrates consist of aldehydes and both silyl enol ethers as well as simple ketones.
Representative selection schemes for Heck coupling, hetero Diels-Alder, and aldol addition catalysts are shown in Figs. 57A, 57B, and 57C, respectively. The stringency of these selections can be increased between rounds of selection by decreasing reaction times, lowering reaction temperatures, or using less activated substrates (for example, less electron poor aryl chlorides (Littke et al. J. Am. Chem. Soc. 2001, 123, 6989-7000) or simple ketones instead of silyl enol ethers).
[00235] Evolving Non-Natural Polymers: Diversification and Selecting for Stereospecificity 78 of 88 WO 02/074929 PCT/US02/08546 00 O [00236] Following each round of selection, active library members are amplified by PCR with the non-natural nucleotides and subjected to additional rounds of selection to enrich the library for desired catalysts. These libraries are truly evolved by introducing a diversification step C before each round of selection. Libraries are diversified by random mutagenesis using errorprone PCR (Caldwell et al. PCR Methods Applic. 1992, 2, 28-33) or by recombination using modified DNA shuffling methods that recombine small, non-homologous nucleic acid fragments.
(Cl Because error-prone PCR is inherently less efficient than normal PCR, error-prone PCR 0 diversification will be conducted with only natural dATP, dTTP, dCTP, and dGTP and using primers that lack chemical handles or biotin groups. The resulting mutagenized products are Sthen subjected to PCR translation into non-natural nucleic acid polymers using standard PCR Sreactions containing the non-natural nucleotide(s), the biotinylated primer, and the amino- or thiol-terminated primer.
[00237] In addition to simply evolving active catalysts, the in vitro selections described above are used to evolve non-natural polymer libraries in powerful directions difficult to achieve using other catalyst discovery approaches. An enabling feature of these selections is the ability to select either for library members that are biotinylated or for members that are not biotinylated.
Substrate specificity among catalysts can therefore be evolved by selecting for active catalysts in the presence of the desired substrate and then selecting in the same pot for inactive catalysts in the presence of one or more undesired substrates. If the desired and undesired substrates differ by the configuration at one or more stereocenters, enantioselective or diastereoselective catalysts can emerge from rounds of selection. Similarly, metal selectivity can be evolved by selecting for active catalysts in the presence of desired metals and selecting for inactive catalysts in the presence of undesired metals. Conversely, catalysts with broad substrate tolerance can be evolved by varying substrate structures between successive rounds of selection.
[00238] Finally, the observations of sequence-specific DNA-templated synthesis in DMF and
CH
2 C1 2 suggests that DNA-tetralkylammonium cation complexes can form base-paired structures in organic solvents. This finding raises the possibility of evolving our non-natural nucleic acid catalysts in organic solvents using slightly modified versions of the selections described above. The actual bond forming and bond cleavage selection reactions will be conducted in organic solvents, the crude reactions will be ethanol precipitated to remove the tetraalkylammonium cations, and the immobilized avidin separation of biotinylated and non- 79 of 88 WO 02/074929 PCT/US02/08546 00 0 O biotinylated library members in aqueous solution will be performed. PCR amplification of selected members will then take place as described above. The successful evolution of reaction catalysts that function in organic solvents would expand considerably both the scope of reactions CM that can be catalyzed and the utility of the resulting evolved non-natural polymer catalysts.
[00239] Characterizing Evolved Non-Natural Polymers: Libraries subjected to several rounds of evolution are characterized for their ability to catalyze the reactions of interest both as C1 pools 'of mixed sequences or as individual library members. Individual members are extricated Sfrom evolved pools by ligating PCR amplified sequences into DNA vectors, transforming dilute C'l solutions of ligated vectors into competent bacterial cells, and picking single colonies of 00 0transformants. Assays on pools or individual sequences are conducted both in the single turnover format and in a true multiple turnover catalytic format. For the single turnover assays, the rate at which substrate-linked bond formation catalysts effect their own biotinylation in the presence of free biotinylated substrate will be measured, or the rate at which biotinylated bond breakage catalysts effect the loss of their biotin groups. Multiple turnover assays are conducted by incubating evolved catalysts with small molecule versions of substrates and analyzing the rate of product formation by tic, NMR, mass spectrometry, HPLC, or spectrophotometry.
[00240] Once multiple turnover catalysts are evolved and verified by these methods, detailed mechanistic studies can be conducted on the catalysts. The DNA sequences corresponding to the catalysts are revealed by sequencing PCR products or DNA vectors containing the templates of active catalysts. Metal preferences are evaluated by metalating catalysts with a wide variety of metal cations and measuring the resulting changes in activity. The substrate specificity and stereoselectivity of these catalysts are assessed by measuring the rates of turnover of a series of substrate analogs. Diastereoselectivities and enantioselectivities of product formation are revealed by comparing reaction products with those of known stereochemistry. Previous studies suggest that active sites buried within large chiral environments often possess high degrees of stereoselectivity. For example, peptide-based catalysts generated in combinatorial approaches have demonstrated poor to excellent stereoselectivities that correlate with the size of the peptide ligand (Jarvo et al. J. Am. Chem. Soc. 1999, 121, 11638-11643) while RNA-based catalysts and antibody-based catalysts frequently demonstrate excellent stereoselectivities (Jischke et al. Curr.
Opin. Chem. Biol. 2000, 4, 257-262; Seelig et al. Angew. Chem. Int. Ed. Engl. 2000, 39, 4576- 4579; Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-93; Barbas et al. Science 1997, 278, 2085of 88 WO 02/074929 PCT/US02/08546 00 O 92; Zhong et al. Angew. Chem. Int. Ed. Engl. 1999, 38, 3738-3741; Zhong et al. J. Am. Chem.
Soc. 1997, 119, 8131-8132; List et al. Org. Lett. 1999, 1, 59-61) The direct selections for substrate stereoselectivity described above should further enhance this property among evolved CC catalysts.
[00241] Structure-function studies on evolved catalysts are greatly facilitated by the ease of automated DNA synthesis. Site-specific structural modifications are introduced by synthesizing Cr DNA sequences corresponding to "mutated" catalysts in which bases of interest are changed to Sother bases. Changing the non-natural bases in a catalyst to a natural base to C or A* to G) r, and assaying the resulting mutants may identify the chemically important metal-binding sites in 00 each catalyst. The minimal polymer required for efficient catalysis are determined by synthesizing and assaying progressively truncated versions of active catalysts. Finally, the threedimensional structures of the most interesting evolved catalysts complexed with metals are solved in collaboration with local macromolecular NMR spectroscopists or X-ray crystallographers.
[00242] Throughout this specification and the claims, unless the context requires otherwise, the word "comprise" and its variations, such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that such art forms part of the common general knowledge in Australia.
81 of 88
Claims (29)
- 2. The method of claim 1, wherein a portion of the one or more chemical compounds contain an anti-codon comprising a nucleotide sequence which hybridizes with one or more nucleic acid templates.
- 3. The method of claim 1, wherein a library of more than one compound is synthesized, the method further comprising: probing the library to detect a library member comprising a reaction product of the reactive units displaying a desired property; detecting structural information about the reaction product; and iterating the method with one or more additional species to produce a new generation of reaction products, at least some of which display the desired property. 3. The method of claim 1, wherein the chemical compound syntheiszed is a compound other than a nucleic acid or nucleic acid analog.
- 4. The method of claim 1, wherein the chemical compound is an unnatural polymer. The method of claim 1, wherein the chemical compound is a small molecule. 82 of 88 WO 02/074929 PCT/US02/08546 00 6. The method of claim 1, wherein the chemical compound is a small molecule and the step 0 0 of contacting comprisies contacting with two or more transfer units in a sequential manner. c- 7. The method of claim 1, wherein the template is a nucleic acid template.
- 8. The method of claim 1, wherein the template comprises DNA or RNA. O 9. The method of claim 1, wherein the template comprises DNA. 0 0
- 10. A library comprising one or more chemical compounds wherein each of the chemical Scompounds is bonded to an amplifiable template whose nucleotide sequence is informative of the structure of the chemical compounds.
- 11. The library of claim 10, wherein the library of chemical compounds comprises a library of small molecules.
- 12. A method for the synthesis of a library of chemical compounds, the method comprising the steps of: providing one or more templates optionally associated with one or more reactive units; contacting the one or more templates simultaneously or sequentially with one or more transfer units comprising anti-codon units associated with one or more reactive units under condition suitable for hybridization of the anti-codons with the template and reaction of the reactive units to produce a plurality of different library members.
- 13. The method of claim 12, wherein a portion of the compounds contain an anti-codon comprising a nucleotide sequence which hybridizes with one or more nucleic acid templates.
- 14. The method of claim 12, the method further comprising: probing the library to detect a library member comprising a reaction product of the reactive units displaying a desired property; detecting structural information about the reaction product; and 83 of 88 WO 02/074929 PCT/US02/08546 00 iterating the method with one or more additional species to produce a new generation of 0 0 reaction products, at least some of which display the desired property. c. 15. The method of claim 14, wherein the desired property comprises binding to a target r protein. r> 16. The method of claim 14, wherein the desired property comprises catalyzing a chemical 0 reaction. 0 0
- 17. The method of claim 14, further comprising the step of isolating the one or chemical compounds.
- 18. The method of claim 14, wherein the chemical compounds synthesized are small molecules.
- 19. A method for the synthesis of one or more unnatural polymers, the method comprising steps of: providing one or more nucleic acid templates; contacting one or more transfer units with the one or more nucleic acid template under conditions to allow for hybridization and reaction to form bonds between adjacent monomer units lined up along the template. The method of claim 19, wherein the method further comprises the step of isolating the unnatural polymer.
- 21. The method of claim 19, wherein a portion of the unnatural polymers contain an anti- codon comprising a nucleotide sequence which hybridizes with one or more nucleic acid templates.
- 22. The method of claim 19, the method further comprising: 84 of 88 WO 02/074929 PCT/US02/08546 probing the library to detect a library member comprising a reaction product of the reactive units displaying a desired property; detecting structural information about the reaction product; and iterating the method with one or more additional species to produce a new generation of reaction products, at least some of which display the desired property.
- 24. The method of claim 23, wherein the desired property comprises catalyzing a chemical reaction. The method of claim 12 or 19, wherein the nucleic acid template is DNA.
- 26. The method of claim 12 or 19, wherein the nucleic acid template is single-stranded.
- 27. The method of claim 12 or 19, wherein the nucleic acid template is double-stranded.
- 28. The method of claim 12 or 19, wherein the nucleic acid template is selected from the group consisting of DNA, RNA, a hybrid of DNA and RNA, a derivative of DNA, and a derivative of RNA.
- 29. The method of claim 12 or 19, wherein the anti-codons comprise 2, 3, 4, 5, 6, 7, 8, 9, or bases. The method of claim 12 or 19, wherein the anti-codon does not allow frame-shifting.
- 31. The method of claim 12 or 19, wherein the anti-codon is selected from the group consisting of DNA, RNA, a hybrid of DNA and RNA, a derivative of DNA, and a derivative of RNA.
- 32. The method of claim 12 or 19, wherein the anticodon is associated with the monomer unit through a covalent bond. of 88 WO 02/074929 PCT/US02/08546 00 33. A method of evolving a library of compounds, the method comprising steps of: Sisolating a compound with a desired activity attached to a nucleic acid template that encoded its synthesis; h. mutating and amplifying the nucleic acid template; and Ssynthesizing a new library of related compounds using a mutated and amplified nucleic acid template. 0 34. The method of claim 33 further comprising steps of: 0 assaying compounds for desired activity; and 0 0 repeating steps in claim 33 based on template of polymer with desired activity. The method of claim 34 wherein the desired activity is selected from the groups consisting of catalytic activity and binding activity.
- 36. The method of claim 34 wherein the step of mutating and amplifying is performed using the polymerase chain reaction.
- 37. The method of claim 34 wherein the step of mutating and amplifying is performed using error-prone PCR.
- 38. The method of claim 34 wherein the step of mutating and amplifying is performed using in vitro homologous recombination (DNA shuffling).
- 39. A library of chemical compounds comprising one or more chemical compounds wherein each of the chemical compounds is bonded to an amplifiable template whose nucleotide sequence is informative of the structure of the chemical compounds, and wherein the library is synthesized according to the method of claim 1, 12 or 19. The library of claim 39, wherein the library is a library of small molecules or unnatural polymers. 86 of 88 WO 02/074929 PCT/US02/08546 00 41. A kit comprising one or more nucleic acid templates and one or more transfer units. O
- 42. The kit of claim 41 further comprising buffers, heat-stable DNA polymerase, nucleotides, c- and restriction endonucleases.
- 43. The kit of claim 41 wherein the nucleic acid template is associated with a small molecule scaffold.
- 44. The kit of claim 41 comprising a plurality of nucleic acid templates. 00 The kit of claim 41 wherein the transfer units comprise monomer units.
- 46. The kit of claim 41 wherein the transfer units comprise reactants to be used in modifying a small molecule scaffold. 87 of 88
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2008200027A AU2008200027B2 (en) | 2001-03-19 | 2008-01-03 | Evolving new molecular function |
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US27708101P | 2001-03-19 | 2001-03-19 | |
US27709401P | 2001-03-19 | 2001-03-19 | |
US60/277,081 | 2001-03-19 | ||
US60/277,094 | 2001-03-19 | ||
US30669101P | 2001-07-20 | 2001-07-20 | |
US60/306,691 | 2001-07-20 | ||
US10/101,030 | 2002-03-19 | ||
AU2002306777A AU2002306777C1 (en) | 2001-03-19 | 2002-03-19 | Evolving new molecular function |
US10/101,030 US7070928B2 (en) | 2001-03-19 | 2002-03-19 | Evolving new molecular function |
PCT/US2002/008546 WO2002074929A2 (en) | 2001-03-19 | 2002-03-19 | Evolving new molecular function |
AU2008200027A AU2008200027B2 (en) | 2001-03-19 | 2008-01-03 | Evolving new molecular function |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2002306777A Division AU2002306777C1 (en) | 2001-03-19 | 2002-03-19 | Evolving new molecular function |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2008200027A1 true AU2008200027A1 (en) | 2008-01-31 |
AU2008200027B2 AU2008200027B2 (en) | 2010-05-27 |
Family
ID=27493169
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2002306777A Ceased AU2002306777C1 (en) | 2001-03-19 | 2002-03-19 | Evolving new molecular function |
AU2008200027A Ceased AU2008200027B2 (en) | 2001-03-19 | 2008-01-03 | Evolving new molecular function |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2002306777A Ceased AU2002306777C1 (en) | 2001-03-19 | 2002-03-19 | Evolving new molecular function |
Country Status (12)
Country | Link |
---|---|
US (7) | US7070928B2 (en) |
EP (3) | EP1832567A3 (en) |
JP (4) | JP4128453B2 (en) |
AT (1) | ATE335754T1 (en) |
AU (2) | AU2002306777C1 (en) |
CA (1) | CA2441820C (en) |
DE (1) | DE60213826T3 (en) |
DK (1) | DK1423400T4 (en) |
ES (1) | ES2271306T5 (en) |
IL (4) | IL158000A0 (en) |
PT (1) | PT1423400E (en) |
WO (1) | WO2002074929A2 (en) |
Families Citing this family (121)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7666588B2 (en) | 2001-03-02 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy |
US20040121335A1 (en) | 2002-12-06 | 2004-06-24 | Ecker David J. | Methods for rapid detection and identification of bioagents associated with host versus graft and graft versus host rejections |
US20030027135A1 (en) | 2001-03-02 | 2003-02-06 | Ecker David J. | Method for rapid detection and identification of bioagents |
US7226739B2 (en) | 2001-03-02 | 2007-06-05 | Isis Pharmaceuticals, Inc | Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations |
PT1423400E (en) * | 2001-03-19 | 2006-12-29 | Harvard College | Evolving new molecular function |
WO2002102820A1 (en) | 2001-06-20 | 2002-12-27 | Nuevolution A/S | Nucleoside derivatives for library preparation |
EP1905829B1 (en) * | 2001-06-20 | 2016-02-17 | Nuevolution A/S | Templated molecules and methods for using such molecules |
US8073627B2 (en) * | 2001-06-26 | 2011-12-06 | Ibis Biosciences, Inc. | System for indentification of pathogens |
US7217510B2 (en) | 2001-06-26 | 2007-05-15 | Isis Pharmaceuticals, Inc. | Methods for providing bacterial bioagent characterizing information |
DE10147074A1 (en) * | 2001-09-25 | 2003-05-08 | Beru Ag | Method for operating a multi-stage electric heater consisting of several heating elements |
DE20200926U1 (en) * | 2002-01-23 | 2002-04-18 | Hegenscheidt-MFD GmbH & Co. KG, 41812 Erkelenz | Deep rolling device of a deep rolling machine for crankshafts |
EA006965B1 (en) | 2002-03-08 | 2006-06-30 | Айдгенёссише Текнише Хохшуле Цюрих | Encoded self-assembling chemical libraries |
EP1487851A2 (en) * | 2002-03-15 | 2004-12-22 | Nuevolution A/S | A building block capable of functional entity transfer to nucleophil |
WO2003078050A2 (en) * | 2002-03-15 | 2003-09-25 | Nuevolution A/S | A building block forming a c-c bond upon reaction |
WO2003078446A2 (en) * | 2002-03-15 | 2003-09-25 | Nuevolution A/S | A building block forming a c-c or a c-hetero atom bond upon reaction |
DK1487978T3 (en) * | 2002-03-15 | 2009-03-23 | Nuevolution As | Improved method for synthesis of template molecules |
EP1539980B1 (en) | 2002-08-01 | 2016-02-17 | Nuevolution A/S | Library of complexes comprising small non-peptide molecules and double-stranded oligonucleotides identifying the molecules |
US7491494B2 (en) * | 2002-08-19 | 2009-02-17 | President And Fellows Of Harvard College | Evolving new molecular function |
WO2004024929A2 (en) * | 2002-09-12 | 2004-03-25 | Nuevolution A/S | Proximity-aided synthesis of templated molecules |
EP1545762A2 (en) * | 2002-09-27 | 2005-06-29 | Carlsberg A/S | Spatially encoded polymer matrix |
AU2011226815B2 (en) * | 2002-10-30 | 2014-09-25 | Nuevolution A/S | Enzymatic encoding |
CN106337046B (en) * | 2002-10-30 | 2021-03-23 | 纽韦卢森公司 | Method for synthesizing bifunctional complexes |
JP2006516193A (en) | 2002-12-06 | 2006-06-29 | アイシス・ファーマシューティカルス・インコーポレーテッド | Rapid identification of pathogens in humans and animals |
EP2175019A3 (en) | 2002-12-19 | 2011-04-06 | Nuevolution A/S | Quasirandom structure and function guided synthesis methods |
EP1597395A2 (en) | 2003-02-21 | 2005-11-23 | Nuevolution A/S | Method for producing second-generation library |
WO2004074501A2 (en) * | 2003-02-21 | 2004-09-02 | Nuevolution A/S | A method for obtaining structural information about an encoded molecule |
EP1608748B1 (en) * | 2003-03-20 | 2009-03-04 | Nuevolution A/S | Ligational encoding of small molecules |
US8017323B2 (en) * | 2003-03-26 | 2011-09-13 | President And Fellows Of Harvard College | Free reactant use in nucleic acid-templated synthesis |
US8046171B2 (en) | 2003-04-18 | 2011-10-25 | Ibis Biosciences, Inc. | Methods and apparatus for genetic evaluation |
US8057993B2 (en) | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
EP1641730B1 (en) * | 2003-05-08 | 2014-07-30 | Valent Technologies, LLC | Recombined molecules and preparation thereof |
US7964343B2 (en) | 2003-05-13 | 2011-06-21 | Ibis Biosciences, Inc. | Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
US8158354B2 (en) | 2003-05-13 | 2012-04-17 | Ibis Biosciences, Inc. | Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture |
WO2004110964A2 (en) * | 2003-06-16 | 2004-12-23 | Nuevolution A/S | Encoded molecules by translation (emt) |
WO2005003778A2 (en) * | 2003-07-02 | 2005-01-13 | Nuevolution A/S | A method for identifying a synthetic molecule having affinity towards a target |
US20120122101A1 (en) | 2003-09-11 | 2012-05-17 | Rangarajan Sampath | Compositions for use in identification of bacteria |
US8097416B2 (en) | 2003-09-11 | 2012-01-17 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
US8546082B2 (en) | 2003-09-11 | 2013-10-01 | Ibis Biosciences, Inc. | Methods for identification of sepsis-causing bacteria |
DK1670939T3 (en) * | 2003-09-18 | 2010-03-01 | Nuevolution As | Method for obtaining structural information on a coded molecule and method for selecting compounds |
RU2470077C2 (en) | 2003-12-17 | 2012-12-20 | ГЛЭКСОСМИТКЛАЙН ЭлЭлСи | Biologically active compound containing coding oligonucleotide (versions), method for synthesis thereof, library of compounds (versions), method for synthesis thereof, and method of searching for compound bound with biological target (versions) |
US7972994B2 (en) | 2003-12-17 | 2011-07-05 | Glaxosmithkline Llc | Methods for synthesis of encoded libraries |
EP1723255B1 (en) * | 2004-02-17 | 2010-12-29 | Nuevolution A/S | Method for enrichment involving elimination by mismatch hybridisation |
US7666592B2 (en) | 2004-02-18 | 2010-02-23 | Ibis Biosciences, Inc. | Methods for concurrent identification and quantification of an unknown bioagent |
US8119336B2 (en) | 2004-03-03 | 2012-02-21 | Ibis Biosciences, Inc. | Compositions for use in identification of alphaviruses |
WO2005090566A2 (en) * | 2004-03-22 | 2005-09-29 | Nuevolution A/S | Ligational encoding using building block oligonucleotides |
JP4810533B2 (en) | 2004-05-24 | 2011-11-09 | アイビス バイオサイエンシズ インコーポレイティッド | Mass spectrometry using selective ion filtration by digital thresholding. |
US20050266411A1 (en) | 2004-05-25 | 2005-12-01 | Hofstadler Steven A | Methods for rapid forensic analysis of mitochondrial DNA |
CA2565967A1 (en) | 2004-06-10 | 2005-12-29 | Perkinelmer Las, Inc. | Multiplexing assays for analyte detection |
US7811753B2 (en) | 2004-07-14 | 2010-10-12 | Ibis Biosciences, Inc. | Methods for repairing degraded DNA |
WO2006135400A2 (en) | 2004-08-24 | 2006-12-21 | Isis Pharmaceuticals, Inc. | Methods for rapid identification of recombinant organisms |
US8202823B2 (en) | 2004-11-08 | 2012-06-19 | Vipergen Aps | Structural nucleic acid guided chemical synthesis |
WO2006053571A2 (en) | 2004-11-22 | 2006-05-26 | Peter Birk Rasmussen | Template directed split and mix systhesis of small molecule libraries |
CA2600184A1 (en) | 2005-03-03 | 2006-09-08 | Isis Pharmaceuticals, Inc. | Compositions for use in identification of adventitious viruses |
US8084207B2 (en) | 2005-03-03 | 2011-12-27 | Ibis Bioscience, Inc. | Compositions for use in identification of papillomavirus |
ATE483825T1 (en) * | 2005-04-13 | 2010-10-15 | Ibis Biosciences Inc | COMPOSITIONS FOR IDENTIFYING ADENOVIRUSES |
WO2007008276A2 (en) * | 2005-05-03 | 2007-01-18 | Ensemble Discovery Corporation | Turnover probes and use thereof for nucleic acid detection |
CN101248189B (en) * | 2005-05-26 | 2013-05-01 | 通信改革公司 | Biodetection by nucleic acid-templated chemistry |
US20090203530A1 (en) * | 2005-06-07 | 2009-08-13 | Liu David R | Polymer evolution via templated synthesis |
JP5508711B2 (en) | 2005-06-09 | 2014-06-04 | グラクソスミスクライン・リミテッド・ライアビリティ・カンパニー | Method for the synthesis of coded libraries |
US8183178B2 (en) * | 2005-06-17 | 2012-05-22 | President And Fellows Of Harvard College | Iterated branching reaction pathways via nucleic acid-mediated chemistry |
US20090035824A1 (en) * | 2005-06-17 | 2009-02-05 | Liu David R | Nucleic acid-templated chemistry in organic solvents |
WO2007011722A2 (en) | 2005-07-15 | 2007-01-25 | President And Fellows Of Harvard College | Reaction discovery system |
US8026084B2 (en) | 2005-07-21 | 2011-09-27 | Ibis Biosciences, Inc. | Methods for rapid identification and quantitation of nucleic acid variants |
DE602005003374T2 (en) * | 2005-10-17 | 2008-09-11 | Pls-Design Gmbh | Method for the determination of an unknown PNS sequence and its uses |
AU2006309096B2 (en) | 2005-10-28 | 2013-07-04 | Glaxosmithkline Llc | Methods for identifying compounds of interest using encoded libraries |
SI2336315T1 (en) | 2005-12-01 | 2017-12-29 | Nuevolution A/S | Enzymatic encoding methods for efficient synthesis of large libraries |
EP2021470A1 (en) | 2006-05-03 | 2009-02-11 | Vipergen APS | A method for preparing compounds by nucleic acid directed synthesis |
US9149473B2 (en) | 2006-09-14 | 2015-10-06 | Ibis Biosciences, Inc. | Targeted whole genome amplification method for identification of pathogens |
US20100159455A1 (en) * | 2006-09-18 | 2010-06-24 | Ensemble Discovery | Receptor family profiling |
AU2007314516A1 (en) * | 2006-09-28 | 2008-05-08 | Ensemble Discovery Corporation | Compositions and methods for biodetection by nucleic acid-templated chemistry |
WO2008104002A2 (en) | 2007-02-23 | 2008-08-28 | Ibis Biosciences, Inc. | Methods for rapid forensic dna analysis |
US9598724B2 (en) | 2007-06-01 | 2017-03-21 | Ibis Biosciences, Inc. | Methods and compositions for multiple displacement amplification of nucleic acids |
US20100159446A1 (en) * | 2007-07-27 | 2010-06-24 | Haff Lawrence A | Detection Assays and Use Thereof |
US20130217588A1 (en) * | 2008-01-18 | 2013-08-22 | The Johns Hopkins University | Methods for Identifying Eubacteria |
EP2350031A2 (en) * | 2008-08-20 | 2011-08-03 | Ensemble Therapeutics Corporation | Macrocyclic compounds for inhibition of tumor necrosis factor alpha |
JP2012502911A (en) * | 2008-09-14 | 2012-02-02 | インサイター インコーポレイテッド | Nanoscale molecular synthesis |
EP2344893B1 (en) | 2008-09-16 | 2014-10-15 | Ibis Biosciences, Inc. | Microplate handling systems and methods |
US8148163B2 (en) | 2008-09-16 | 2012-04-03 | Ibis Biosciences, Inc. | Sample processing units, systems, and related methods |
US8550694B2 (en) | 2008-09-16 | 2013-10-08 | Ibis Biosciences, Inc. | Mixing cartridges, mixing stations, and related kits, systems, and methods |
EP2396803A4 (en) | 2009-02-12 | 2016-10-26 | Ibis Biosciences Inc | Ionization probe assemblies |
MX2011008563A (en) * | 2009-02-13 | 2012-06-19 | Chem Inc X | Methods of creating and screening dna-encoded libraries. |
DK2446033T3 (en) | 2009-06-22 | 2014-01-27 | Univ Strasbourg | PROCEDURE FOR MANUFACTURING A ADDUCT |
EP2454000A4 (en) | 2009-07-17 | 2016-08-10 | Ibis Biosciences Inc | Systems for bioagent identification |
US8950604B2 (en) | 2009-07-17 | 2015-02-10 | Ibis Biosciences, Inc. | Lift and mount apparatus |
EP3225695A1 (en) | 2009-10-15 | 2017-10-04 | Ibis Biosciences, Inc. | Multiple displacement amplification |
ES2713873T3 (en) | 2010-04-16 | 2019-05-24 | Nuevolution As | Bifunctional complexes and methods for making and using such complexes |
DK3399035T3 (en) | 2010-09-27 | 2021-05-17 | Vipergen Aps | Procedure for making an enriched library |
JP6040227B2 (en) | 2011-05-19 | 2016-12-07 | アジーナ バイオサイエンス, インコーポレイテッド | Products and processes for multiplex nucleic acid identification |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
CA2848023C (en) | 2011-09-07 | 2022-03-15 | X-Chem, Inc. | Methods for tagging dna-encoded libraries |
EA201992285A1 (en) | 2012-07-13 | 2020-05-31 | Икс-Чем, Инк. | DNA-CODED LIBRARIES CONTAINING LINKS OF CODING OLIGONUCLEOTIDES NOT AVAILABLE FOR READING BY POLYMERASES |
CN105051270B (en) | 2013-03-15 | 2019-05-07 | 代表亚利桑那大学的亚利桑那校董会 | Biosensor microarray constitutes and method |
US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | President And Fellows Of Harvard College | Methods for identifying a target site of a cas9 nuclease |
US9340800B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | Extended DNA-sensing GRNAS |
GB201322692D0 (en) | 2013-12-20 | 2014-02-05 | Philochem Ag | Production of encoded chemical libraries |
AU2015298571B2 (en) | 2014-07-30 | 2020-09-03 | President And Fellows Of Harvard College | Cas9 proteins including ligand-dependent inteins |
US10040048B1 (en) | 2014-09-25 | 2018-08-07 | Synthego Corporation | Automated modular system and method for production of biopolymers |
WO2016074683A1 (en) | 2014-11-11 | 2016-05-19 | Lundorf Pedersen Materials Aps | Method for identification of molecules with desired characteristics |
JP6997623B2 (en) * | 2014-12-12 | 2022-02-04 | エム. ウルフ、トッド | Compositions and Methods for Editing Intracellular Nucleic Acids Utilizing Oligonucleotides |
CA2983224A1 (en) | 2015-04-24 | 2016-10-27 | Agena Bioscience, Inc. | Multiplexed method for the identification and quantitation of minor alleles and polymorphisms |
US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
CN109312492B (en) | 2016-06-16 | 2022-10-04 | 哈斯达克科学公司 | Combinatorial synthesis of oligonucleotide directed and recorded coded probe molecules |
IL264565B2 (en) | 2016-08-03 | 2024-07-01 | Harvard College | Adenosine nucleobase editors and uses thereof |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
WO2018081534A1 (en) | 2016-10-28 | 2018-05-03 | President And Fellows Of Harvard College | Assay for exo-site binding molecules |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
WO2018165504A1 (en) | 2017-03-09 | 2018-09-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
KR20190127797A (en) | 2017-03-10 | 2019-11-13 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Cytosine to Guanine Base Editing Agent |
WO2018204420A1 (en) | 2017-05-02 | 2018-11-08 | Haystack Sciences Corporation | Molecules for verifying oligonucleotide directed combinatorial synthesis and methods of making and using the same |
WO2018209320A1 (en) | 2017-05-12 | 2018-11-15 | President And Fellows Of Harvard College | Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
EP3681637A4 (en) * | 2017-09-11 | 2021-06-02 | Synthego Corporation | Biopolymer synthesis system and method |
JP2021500036A (en) | 2017-10-16 | 2021-01-07 | ザ ブロード インスティテュート, インコーポレーテッドThe Broad Institute, Inc. | Use of adenosine base editing factors |
WO2019168654A2 (en) | 2018-02-09 | 2019-09-06 | President And Fellows Of Harvard College | Dna-templated macrocycle library |
US20220042079A1 (en) | 2018-12-20 | 2022-02-10 | Sergey N. Krylov | Binder Selection Using Capollary Electrophoresis |
AU2020211270A1 (en) | 2019-01-22 | 2021-07-15 | Vipergen Aps | A method for screening of an in vitro display library within a cell |
WO2020191153A2 (en) | 2019-03-19 | 2020-09-24 | The Broad Institute, Inc. | Methods and compositions for editing nucleotide sequences |
GB2614813A (en) | 2020-05-08 | 2023-07-19 | Harvard College | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
WO2023164224A1 (en) * | 2022-02-28 | 2023-08-31 | The Board Of Trustees Of The Leland Stanford Junior University | Functionalized nanoclusters and their use in treating bacterial infections |
Family Cites Families (89)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US277081A (en) | 1883-05-08 | Scalding napped hats and sizing or felting hat and other fabrics | ||
US4194550A (en) * | 1976-01-26 | 1980-03-25 | Insulating Shade (Limited Partnership) | Apparatus for insulating against conductive, convective and radiant heat transmission |
US4863857A (en) | 1985-03-01 | 1989-09-05 | Board Of Regents, The University Of Texas System | Polypeptide complementary to peptides or proteins having an amino acid sequence or nucleotide coding sequence at least partially known |
US5506337A (en) | 1985-03-15 | 1996-04-09 | Antivirals Inc. | Morpholino-subunit combinatorial library and method |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
US4703034A (en) * | 1986-04-28 | 1987-10-27 | Merck & Co., Inc. | Novel cyclic tetrapeptide |
US5449602A (en) * | 1988-01-13 | 1995-09-12 | Amoco Corporation | Template-directed photoligation |
US5162218A (en) | 1988-11-18 | 1992-11-10 | The Regents Of The University Of California | Conjugated polypeptides and methods for their preparation |
US6680192B1 (en) | 1989-05-16 | 2004-01-20 | Scripps Research Institute | Method for producing polymers having a preselected activity |
US6291160B1 (en) | 1989-05-16 | 2001-09-18 | Scripps Research Institute | Method for producing polymers having a preselected activity |
US6291161B1 (en) | 1989-05-16 | 2001-09-18 | Scripps Research Institute | Method for tapping the immunological repertiore |
EP0494955B1 (en) | 1989-10-05 | 1998-07-15 | Optein, Inc. | Cell-free synthesis and isolation of novel genes and polypeptides |
US5660985A (en) | 1990-06-11 | 1997-08-26 | Nexstar Pharmaceuticals, Inc. | High affinity nucleic acid ligands containing modified nucleotides |
US5637682A (en) * | 1990-06-11 | 1997-06-10 | Nexstar Pharmaceuticals, Inc. | High-affinity oligonucleotide ligands to the tachykinin substance P |
US5723289A (en) | 1990-06-11 | 1998-03-03 | Nexstar Pharmaceuticals, Inc. | Parallel selex |
WO1992002536A1 (en) | 1990-08-02 | 1992-02-20 | The Regents Of The University Of Colorado | Systematic polypeptide evolution by reverse translation |
US20020038000A1 (en) | 1990-08-02 | 2002-03-28 | Larry Gold | Systematic polypeptide evolution by reverse translation |
US5843701A (en) | 1990-08-02 | 1998-12-01 | Nexstar Pharmaceticals, Inc. | Systematic polypeptide evolution by reverse translation |
US6037120A (en) | 1995-10-12 | 2000-03-14 | Benner; Steven Albert | Recognition of oligonucleotides containing non-standard base pairs |
US6140496A (en) | 1990-10-09 | 2000-10-31 | Benner; Steven Albert | Precursors for deoxyribonucleotides containing non-standard nucleosides |
US5641625A (en) | 1992-05-22 | 1997-06-24 | Isis Pharmaceuticals, Inc. | Cleaving double-stranded DNA with peptide nucleic acids |
US5719262A (en) | 1993-11-22 | 1998-02-17 | Buchardt, Deceased; Ole | Peptide nucleic acids having amino acid side chains |
US5539082A (en) | 1993-04-26 | 1996-07-23 | Nielsen; Peter E. | Peptide nucleic acids |
US5574141A (en) | 1991-08-28 | 1996-11-12 | Boehringer Mannheim Gmbh | Functionalized carrier materials for the simultaneous synthesis and direct labeling of oligonucleotides as primers for template-dependent enzymatic nucleic acid syntheses |
DE69217497T2 (en) | 1991-09-18 | 1997-06-12 | Affymax Tech Nv | METHOD FOR SYNTHESISING THE DIFFERENT COLLECTIONS OF OLIGOMERS |
US5639603A (en) | 1991-09-18 | 1997-06-17 | Affymax Technologies N.V. | Synthesizing and screening molecular diversity |
US5270170A (en) | 1991-10-16 | 1993-12-14 | Affymax Technologies N.V. | Peptide library and screening method |
ATE241426T1 (en) | 1991-11-22 | 2003-06-15 | Affymetrix Inc A Delaware Corp | METHOD FOR PRODUCING POLYMER ARRAYS |
US5573905A (en) * | 1992-03-30 | 1996-11-12 | The Scripps Research Institute | Encoded combinatorial chemical libraries |
US5721099A (en) | 1992-10-01 | 1998-02-24 | Trustees Of Columbia University In The City Of New York | Complex combinatorial chemical libraries encoded with tags |
US5565324A (en) | 1992-10-01 | 1996-10-15 | The Trustees Of Columbia University In The City Of New York | Complex combinatorial chemical libraries encoded with tags |
US6436635B1 (en) | 1992-11-06 | 2002-08-20 | Boston University | Solid phase sequencing of double-stranded nucleic acids |
CA2153387A1 (en) | 1993-01-07 | 1994-07-21 | Hubert Koester | Dna sequencing by mass spectrometry |
US6194144B1 (en) | 1993-01-07 | 2001-02-27 | Sequenom, Inc. | DNA sequencing by mass spectrometry |
US5605798A (en) * | 1993-01-07 | 1997-02-25 | Sequenom, Inc. | DNA diagnostic based on mass spectrometry |
US6074823A (en) | 1993-03-19 | 2000-06-13 | Sequenom, Inc. | DNA sequencing by mass spectrometry via exonuclease degradation |
CA2158642A1 (en) | 1993-03-19 | 1994-09-29 | Hubert Koster | Dna sequencing by mass spectrometry via exonuclease degradation |
US5840485A (en) | 1993-05-27 | 1998-11-24 | Selectide Corporation | Topologically segregated, encoded solid phase libraries |
GB9315847D0 (en) * | 1993-07-30 | 1993-09-15 | Isis Innovation | Tag reagent and assay method |
US5922545A (en) | 1993-10-29 | 1999-07-13 | Affymax Technologies N.V. | In vitro peptide and antibody display libraries |
US5925517A (en) * | 1993-11-12 | 1999-07-20 | The Public Health Research Institute Of The City Of New York, Inc. | Detectably labeled dual conformation oligonucleotide probes, assays and kits |
WO1995014106A2 (en) * | 1993-11-17 | 1995-05-26 | Id Biomedical Corporation | Cycling probe cleavage detection of nucleic acid sequences |
US6127154A (en) | 1994-02-10 | 2000-10-03 | Mosbach; Klaus | Methods for direct synthesis of compounds having complementary structure to a desired molecular entity and use thereof |
US5605793A (en) | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
US20030099945A1 (en) | 1994-09-20 | 2003-05-29 | Invenux, Inc. | Parallel selex |
US6048698A (en) | 1994-09-20 | 2000-04-11 | Nexstar Pharmaceuticals, Inc. | Parallel SELEX™ |
US5597697A (en) | 1994-09-30 | 1997-01-28 | Diamond; Paul | Screening assay for inhibitors and activators of RNA and DNA-dependent nucleic acid polymerases |
GB9524630D0 (en) * | 1994-12-24 | 1996-01-31 | Zeneca Ltd | Chemical compounds |
US5559000A (en) | 1995-01-18 | 1996-09-24 | The Scripps Research Institute | Encoded reaction cassette |
DE19646372C1 (en) | 1995-11-11 | 1997-06-19 | Evotec Biosystems Gmbh | Conjugates of polypeptide and encoding nucleic acid |
US5846839A (en) | 1995-12-22 | 1998-12-08 | Glaxo Group Limited | Methods for hard-tagging an encoded synthetic library |
US5888971A (en) * | 1996-02-20 | 1999-03-30 | Ortho Pharmaceutical Corporation, Inc. | Macrocyclic peptides useful in the treatment of thrombin related disorders |
CA2252048C (en) * | 1996-04-12 | 2008-03-11 | The Public Health Research Institute Of The City Of New York, Inc. | Detection probes, kits and assays |
US5998140A (en) | 1996-07-31 | 1999-12-07 | The Scripps Research Institute | Complex formation between dsDNA and oligomer of cyclic heterocycles |
US6140053A (en) | 1996-11-06 | 2000-10-31 | Sequenom, Inc. | DNA sequencing by mass spectrometry via exonuclease degradation |
US5958703A (en) | 1996-12-03 | 1999-09-28 | Glaxo Group Limited | Use of modified tethers in screening compound libraries |
US6080826A (en) | 1997-01-06 | 2000-06-27 | California Institute Of Technology | Template-directed ring-closing metathesis and ring-opening metathesis polymerization of functionalized dienes |
JP2001509828A (en) * | 1997-01-08 | 2001-07-24 | プロリゴ・エルエルシー | Bioconjugation of macromolecules |
US6261804B1 (en) | 1997-01-21 | 2001-07-17 | The General Hospital Corporation | Selection of proteins using RNA-protein fusions |
IL131016A (en) | 1997-01-21 | 2004-12-15 | Gen Hospital Corp | Selection of proteins using rna-protein fusions |
US6969584B2 (en) | 1997-06-12 | 2005-11-29 | Rigel Pharmaceuticals, Inc. | Combinatorial enzymatic complexes |
US6607878B2 (en) | 1997-10-06 | 2003-08-19 | Stratagene | Collections of uniquely tagged molecules |
AU752220B2 (en) * | 1998-01-27 | 2002-09-12 | British Biocell International Limited | Modified nucleic acid probes and uses thereof |
US5945325A (en) * | 1998-04-20 | 1999-08-31 | California Institute Of Technology | Thermally stable para-nitrobenzyl esterases |
US6287765B1 (en) | 1998-05-20 | 2001-09-11 | Molecular Machines, Inc. | Methods for detecting and identifying single molecules |
US6175001B1 (en) | 1998-10-16 | 2001-01-16 | The Scripps Research Institute | Functionalized pyrimidine nucleosides and nucleotides and DNA's incorporating same |
US7479472B1 (en) * | 1998-10-19 | 2009-01-20 | The Board Of Trustees Of The Leland Stanford Junior University | DNA-templated combinatorial library chemistry |
CA2403209A1 (en) * | 1999-04-08 | 2000-10-19 | Pavel V. Sergeev | Synthesis of biologically active compounds in cells |
CN1378598A (en) * | 1999-08-12 | 2002-11-06 | 麦克西根股份有限公司 | Evolution and use of enzymes for combinathorial and medicinal chemistry |
CA2382545C (en) * | 1999-08-27 | 2013-08-13 | Phylos, Inc. | Methods for encoding and sorting in vitro translated proteins |
US7306904B2 (en) * | 2000-02-18 | 2007-12-11 | Olink Ab | Methods and kits for proximity probing |
US6511809B2 (en) * | 2000-06-13 | 2003-01-28 | E. I. Du Pont De Nemours And Company | Method for the detection of an analyte by means of a nucleic acid reporter |
PT1423400E (en) | 2001-03-19 | 2006-12-29 | Harvard College | Evolving new molecular function |
WO2002102820A1 (en) | 2001-06-20 | 2002-12-27 | Nuevolution A/S | Nucleoside derivatives for library preparation |
US20030143561A1 (en) | 2001-06-20 | 2003-07-31 | Nuevolution A/S | Nucleoside derivatives for library preparation |
EP1905829B1 (en) * | 2001-06-20 | 2016-02-17 | Nuevolution A/S | Templated molecules and methods for using such molecules |
WO2004001042A2 (en) | 2002-06-20 | 2003-12-31 | Nuevolution A/S | Microarrays displaying encoded molecules |
EA006965B1 (en) | 2002-03-08 | 2006-06-30 | Айдгенёссише Текнише Хохшуле Цюрих | Encoded self-assembling chemical libraries |
EP1487848A2 (en) | 2002-03-15 | 2004-12-22 | Nuevolution A/S | A building block forming a c=c double bond upon reaction |
EP1487851A2 (en) | 2002-03-15 | 2004-12-22 | Nuevolution A/S | A building block capable of functional entity transfer to nucleophil |
WO2003078446A2 (en) | 2002-03-15 | 2003-09-25 | Nuevolution A/S | A building block forming a c-c or a c-hetero atom bond upon reaction |
DK1487978T3 (en) | 2002-03-15 | 2009-03-23 | Nuevolution As | Improved method for synthesis of template molecules |
WO2003078050A2 (en) | 2002-03-15 | 2003-09-25 | Nuevolution A/S | A building block forming a c-c bond upon reaction |
US7255991B2 (en) | 2002-03-22 | 2007-08-14 | Emory University | Template-drive processes for synthesizing polymers and components related to such processes |
US7491494B2 (en) | 2002-08-19 | 2009-02-17 | President And Fellows Of Harvard College | Evolving new molecular function |
US8017323B2 (en) * | 2003-03-26 | 2011-09-13 | President And Fellows Of Harvard College | Free reactant use in nucleic acid-templated synthesis |
ATE461279T1 (en) | 2005-06-07 | 2010-04-15 | Harvard College | ORDERED MULTI-STEP SYNTHESIS USING NUCLEIC ACID-MEDIATED CHEMISTRY |
US20090035824A1 (en) | 2005-06-17 | 2009-02-05 | Liu David R | Nucleic acid-templated chemistry in organic solvents |
US8183178B2 (en) | 2005-06-17 | 2012-05-22 | President And Fellows Of Harvard College | Iterated branching reaction pathways via nucleic acid-mediated chemistry |
-
2002
- 2002-03-19 PT PT02753671T patent/PT1423400E/en unknown
- 2002-03-19 JP JP2002574322A patent/JP4128453B2/en not_active Expired - Fee Related
- 2002-03-19 DK DK02753671.3T patent/DK1423400T4/en active
- 2002-03-19 WO PCT/US2002/008546 patent/WO2002074929A2/en active IP Right Grant
- 2002-03-19 CA CA2441820A patent/CA2441820C/en not_active Expired - Fee Related
- 2002-03-19 EP EP06016511A patent/EP1832567A3/en not_active Withdrawn
- 2002-03-19 ES ES02753671T patent/ES2271306T5/en not_active Expired - Lifetime
- 2002-03-19 EP EP10182548A patent/EP2332896A3/en not_active Withdrawn
- 2002-03-19 AU AU2002306777A patent/AU2002306777C1/en not_active Ceased
- 2002-03-19 IL IL15800002A patent/IL158000A0/en unknown
- 2002-03-19 EP EP02753671.3A patent/EP1423400B2/en not_active Expired - Lifetime
- 2002-03-19 AT AT02753671T patent/ATE335754T1/en active
- 2002-03-19 US US10/101,030 patent/US7070928B2/en not_active Expired - Lifetime
- 2002-03-19 DE DE60213826T patent/DE60213826T3/en not_active Expired - Lifetime
-
2003
- 2003-09-18 IL IL158000A patent/IL158000A/en not_active IP Right Cessation
- 2003-12-23 US US10/744,605 patent/US7557068B2/en not_active Expired - Lifetime
-
2004
- 2004-09-24 US US10/949,162 patent/US20050042669A1/en not_active Abandoned
- 2004-09-24 US US10/949,163 patent/US7223545B2/en not_active Expired - Lifetime
-
2005
- 2005-05-31 US US11/141,164 patent/US7998904B2/en not_active Expired - Fee Related
- 2005-05-31 US US11/141,542 patent/US7442160B2/en not_active Expired - Lifetime
-
2008
- 2008-01-03 AU AU2008200027A patent/AU2008200027B2/en not_active Ceased
- 2008-02-22 JP JP2008042250A patent/JP4384229B2/en not_active Expired - Fee Related
-
2009
- 2009-07-17 JP JP2009169425A patent/JP2009232869A/en not_active Withdrawn
-
2010
- 2010-06-23 IL IL206586A patent/IL206586A/en not_active IP Right Cessation
- 2010-06-23 IL IL206585A patent/IL206585A/en not_active IP Right Cessation
-
2011
- 2011-06-30 US US13/173,593 patent/US20120165228A1/en not_active Abandoned
-
2012
- 2012-07-26 JP JP2012165977A patent/JP5509270B2/en not_active Expired - Fee Related
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2002306777B2 (en) | Evolving new molecular function | |
AU2002306777A1 (en) | Evolving new molecular function | |
JP5254934B2 (en) | Evolving new molecular functions | |
JP2005536234A5 (en) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FGA | Letters patent sealed or granted (standard patent) | ||
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |